Agilent 2100 reference

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酵母RNA含量测定

real time RT-qPCR 是检测样本中特定基因表达量的有效方法，包含逆转录步骤和定量PCR步骤。

好的开始是成功的一半，获得高质量的、能正确反应样品中转录情况的RNA，对于其后的定量结果，自然是非常重要的。

Mikael Kubista，教授, R&D TATAA Biocenter主任：RNA质量包含两个方面：样品的纯度和RNA的完整性。

样品纯度可以在样品稀释时得到改善。

我们通常通过Nanodrop测量吸光值来检测纯度。

260/280吸光值的比率应该在2至2.1的范围内，并且是“漂亮”的吸光光谱曲线。

230nm吸收峰应该低。

我们通过利用Experion (Bio-Rad)记录电泳图（electropherogram）检测RNA的质量。

当然，最理想的结果是得到很清晰的18S和28S峰值。

但RNA的完整性是无法改善的，保存样品、固定样品和来源于富含降解酶的器官的样品RNA完整性通常不好。

检测RNA质量的两个原因，一是想要知道某一个实验得到的RNA质量，从而选择适当的RT-qPCR方法，二是识别出研究中的样品的质量是否比正常情况差，避免导致偏差或者不正确的结果。

Jo Vandesompele，根特大学医院（Ghent University Hospital）医学遗传学中心高质量的RNA应该是完整的、不含抑制剂的。

我们习惯利用毛细管凝胶电泳系统来评价28S和 18S条带的比率，过程很快速并且只需要很少量的RNA。

值得注意的是，这个比率是组织或者细胞特异的，所以理想的是你应该将样品与一个同样细胞来源的完整对照比较。

在芯片分析（微阵列研究）中通常都要求进行RNA 质量分析，现在许多定量PCR领域的人们开始考虑RNA质量这个因素。

我们最近在发表的文章说到评价用于PCR分析的RNA的质量尤为重要，这是由于并非每个基因降解水平都相同，严重时甚至可以使你的实验结果出错(Perrez-Novo et al., 2005)。

转录组分析_R

• 基因功能注释 • 基因结构分析 • 鉴定出大量新转录本 • 可变剪接鉴定 • 基因融合鉴定
Genome Res 2010
无参考基因组生物信息分析
• Unigene功能注释 • Unigene的GO分类 • Unigene代谢通路分析 • 预测编码蛋白框（CDS） • Unigene表达差异分析 • Unigene在样品间的差异GO分类和Pathway
有参考基因组序列生物信息分析
• 基因结构优化 • 鉴定基因可变剪接 • 预测新转录本 • SNP 分析 • 基因融合鉴定
有参考基因组序列信息分析流程
Reads 在基因组上的分布
基因结构优化
通过转录组测序鉴定出酵母3’ 和5’ UTR区域 (Nagalakshmi, U. et al.,2008)
富集性分析
De novo reads组装流程
Unigene GO 分类
Unigene COG 功能分类
基因表达差异分析
N1:total tag Number in sample A N2:total tag Number in sample B X :Gene expression level in sample A y :Gene expression level in sample B Reference: Audic S. et al. The significance of
3’-
…-5’
5’-
GTATTTTC GGC AC AG
A
G
A
C
T C
T TG
Cycle 1:按顺序加入反应试剂
合成第一个碱基
清除未反应的碱基和试剂
激发碱基荧光并收集荧光信号
去除阻断基团和荧光基团

安捷伦介绍

保证溯源性
提供校准服务 OTDR: IEC 61746 – OTDR 功率计：IEC 61315 – PM 光谱仪：IEC 62129 – OSA
需要注意的是：现有光通信仪表校准规范并不完善，很多新仪表的校准都还没有国际认可的参考校准规范，这需要我们更多的努力和参与。
Haiyang HU
7
光通信仪表校准市场驱动和挑战
简易测试需要时间短，不需要专用设备
困难测试需要时间长
困难测试需要专用设备
Haiyang HU
8
如何定位校准测试类型？
实例：光谱分析仪校准
简易测试需要时间短，不需要专用设备
困难测试需要时间长
困难测试需要专用设备
高性能校准完成的测试
推荐的测试
简易测试需要时间短，不需要专用设备困难测试需要时间长困难测试需要专用设备
Test cost per device Throughput per day
降低低校准测试成本
• 灵活的测试平台 •保护现有投资 •降低测试成本
提高测试吞吐率
• 自动化集成测试 •提高测试速度 •保证测试可靠性
确保校准的溯源
• 重复稳定测试结果 • 高精度 • 标准的一致性测试 • 统计处理和反馈
光示波器
多波长计
SONET Trib signals
STS & VT Switch Matrix
N2X XM (Sonet/SDH Cards)
传输分析仪
数据协议分析仪
Haiyang HU
内容安排
•光通信常用仪表 •光通信仪表计量方法 •安捷伦在计量上的方案和特点 •安捷伦光通信新仪表及方案
Haiyang HU

生物分析仪

• Agilent 2100 生物分析仪，为电泳及流式细胞分析配备可拆卸的卡套
• Agilent 2100 电泳生物分析仪仅为电泳应用配备可拆卸的卡套
两种仪器配置都包括：
1. 芯片注胶平台
2. 芯片旋涡振荡仪
3. Agilent 2100 专家软件可进行仪器控制和数据分析 (可扩展，以完全支持法规要求)
4
DNA 系列 II 试剂盒
更灵便的核酸分析方案
DNA 芯片用于分析高分辨率的多重 PCR 反应，精确测量每个片段的大小和浓度。该分析的高灵敏度和更宽的线性动态范围使得 PCR 扩增产物中的少量杂质得以检测出来。
DNA 分析的优势
• 高灵敏度 — 使用激光 (LIF) 检测技术来检测低至 0.1ng 的 DNA 片段
高分辨率 PCR 片段分析非常适用于： • 分析小的 PCR 产物 • 多重 PCR 分析 • 分析 RT-PCR 反应 • 小质粒的限制性酶解产物分析 • PCR 反应的优化
荧光
时间 (秒)
对竞争性 PCR，在使 PCR 产物中的全部大小不同的片段良好的分离非常重要。在分
析不同的 PCR 产物的混合物时，DNA 1000 试剂盒优于传统的平板凝胶。除此之外还
适用于多种真核细胞进行分析。
荧光
总事件数: 1719
荧光
荧光
百分率: 42.4 总事件数: 1736
荧光
百分率: 43.8
• 单一平台 — 用流式细胞分析组件升级
您的 Agilent 2100 生物分析仪，使之 Annexin V 可以与磷脂酰丝氨酸 (PS) 结合，磷脂酰丝氨酸是位于完整细胞的膜内面
培养 24h
荧光
百分率: 40.6

Agilent 4395A 网络频谱阻抗分析仪数据手册说明书

Network Measurement1.At relative to 0 dBm output, 50 MHz,23 °C ±5 °C /HP-Agilent-4395A-Spectrum-Network-Analyzer.aspx To buy, sell, rent or trade-in this product please click on the link below:2Network MeasurementcontinuedReceiver CharacteristicsInput characteristicsFrequency range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Hz to 500 MHzInput attenuator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 to 50 dB, 10 dB stepFull scale input level (R, A, B)Attenuator setting (dB) Full scale input level0–10 dBm100 dBm20+10 dBm30+20 dBm40+30 dBm50+30 dBmIF bandwidth (IFBW)2, 10, 30, 100, 300, 1 k, 3 k, 10 k, 30 kHzNote: The IFBW should be set to less than 1/5 of the lowest frequency inthe sweep range.Noise level (referenced to full scale input level, 23 °C ±5 °C)at 10 Hz ≤frequency < 100 Hz, IFBW = 2 Hz . . . . . . . . . . . . . . . . . . . . . .–85 dB (SPC)at 100 Hz ≤frequency < 100 kHz, IFBW = 10 Hz . . . . . . . . . . . . . . . . . . . . . . . . .–85 dBat 100 kHz ≤frequency, IFBW = 10 Hz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-–115 dBInput crosstalkfor input R + 10 dBm input, input attenuator: . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 dBfor input A, B input attenuator: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 dBat < 100 kHzR through A, B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –100 dBothers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –100 dB (SPC)at ≥100 kHzR through A, B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –120 dBothers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –120 dB (SPC)Source crosstalk (for input A, B)(typical for input R)at + 10 dBm output, < 100 kHz, input attenuator: 0 dB . . . . . . . . . . . . . . . . .< –100 dBat + 10 dBm output, ≥100 kHz, input attenuator: 0 dB . . . . . . . . . . . . . . . . .< –120 dBMultiplexer switching impedance changeat input attenuator 0 dB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< 0.5% (SPC)at input attenuator 10 dB and above . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< 0.1% (SPC)Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Type-N femaleImpedance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 ΩnominalReturn lossInput attenuator0 dB10 dB20 dB to 50 dB10 Hz ≤frequency < 100 kHz25 dB125 dB125 dB1100 kHz ≤frequency ≤100 MHz25 dB125 dB25 dB1100 MHz < frequency15 dB115 dB15 dB1Maximum input level+30 dBm (at input attenuator: 40 dB or 50 dB)Maximum safe input level+30 dBm or ±7 Vdc (SPC)1.SPC34Absolute amplitude accuracy (R, A, B)at –10 dBm input, input attenuator:10 dB, frequency ≥100 Hz, IFBW ≤3 kHz, 23 °C ±5 °C, . . . . . . . . . . . .< ±1.5 dB Ratio accuracy (A/R, B/R) (typical for A/B)at –10 dBm input, input attenuator:10 dB, IFBW ≤3 kHz, 23 °C ±5 °C, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±2 dB Dynamic accuracy (A/R, B/R) (typical for A/B)Input level Dynamic accuracy 1(relative to full scale input level)frequency ≥100 Hz 0 dB ≥input level > –10 dB ±0.4 dB –10 dB ≥input Level ≥–60 dB ±0.05 dB –60 dB > input level ≥–80 dB ±0.3 dB –80 dB > input level ≥–100 dB ±3 dB Figure 1-1. Magnitude dynamic accuracy Residual responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –80 dB full scale (SPC)Trace noise (A/R, B/R, A/B)at 50 MHz, both inputs: full scale input level –10 dB, IFBW = 300 Hz . . . . . . . . . . . . . .< 0.005 dB rms (SPC)Stability (A/R, B/R, A/B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±0.01 dB/°C (SPC)Phase characteristics Measurements format . . . . . . . . . . . . . . . . . . .Standard format, expanded phase format Frequency response (deviation from linear phase) (A/R, B/R) (SPC for A/B) at –10 dBm input, input attenuator: 10 dB, IFBW ≤3 kHz, 23 °C ±5 °C . . . . . .< ±12°Dynamic accuracy (A/R, B/R) (SPC for A/B)Input level Dynamic accuracy 1(relative to full scale input level)frequency ≥100 Hz 0 dB ≥input level > –10 dB ±3°–10 dB ≥input Level ≥–60 dB ±0.3°–60 dB > input level ≥–80 dB ±1.8°–80 dB > input level ≥–100 dB ±18°Magnitude Characteristics1.R input level (B input level for A/B) = fullscale input level –10 dB, IFBW = 10 Hz,23 °C ± 5 °CInput level (dB)Magnitude dynamic accuracy D y n a m i c a c c u r a c y (d B )Spec Typical5Figure 1-2. Phase dynamic accuracyTrace noise (A/R, B/R, A/B)at 50 MHz, both inputs:full scale input level –10 dB, IFBW = 300 Hz . . . . . . . . . . . . . . . . .< 0.04°rms (SPC)Stability (A/R, B/R, A/B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±0.1 °/°C (SPC)Group delay characteristicsAperture [Hz] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0.25% to 20% of span AccuracyIn general, the following formula can be used to determine the accuracy, in seconds,of a specific group delay measurement: . . . . . . . . . . . .Phase accuracy (degree)Aperture(Hz) x 360 (degree)Sweep characteristicsSweep type . . . . . . . . . . . . . . . . .Linear frequency, log frequency, power, list frequency Sweep direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Upper direction only Trigger type . . . . . . . . . . . . . . . . . . . . . . . . . .Hold, single, number of groups, continuous Trigger source . . . . . . . . . . . . . . . . . . . .Internal (free run), external, manual, GPIB (bus)Event trigger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .On point, on sweepInput level (dB)Phase dynamic accuracyD y n a m i c a c c u r a c y (d e g r e e )Spec Typical6Frequency characteristics Frequency range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Hz to 500 MHz Frequency readout accuracy . . . . . . . .±((freq readout [Hz ]) x (freq ref accuracy [1]) + RBW [Hz ] + SPAN [Hz ])) [Hz ]where NOP means number of display points NOP -1Frequency reference (Option 4395A-800)Accuracy at 23 °C ±5 °C, referenced to 23 °C . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±5.5 ppm Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±2.5 ppm/year (SPC) Initial achievable accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±1.0 ppm (SPC) Temperature stability at 23 °C ±5 °C, referenced to 23 °C . . . . . . . . . . . . . . . . . . . . . . . . .< ±2 ppm (SPC) Precision frequency reference (Option 4395A-1D5) Accuracy at 0 °C to 40 °C, referenced to 23 °C . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±0.13 ppm Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±0.l ppm/year (SPC)Initial achievable accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±0.02 ppm (SPC)Temperature stability at 0 °C to 40 °C, referenced to 23 °C . . . . . . . . . . . . . . . . . . . . . . . .< ±0.01 ppm (SPC)Resolution bandwidth (RBW)Range 3 dB RBW at span > 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Hz to 1 MHz, 1-3 step 3 dB RBW at span = 0 . . . . . . . . . . . .3 k, 5 k, 10 k, 20 k, 40 k, 100 k, 200 k, 400 k, 800 k, 1.5 M, 3 M, 5 MHz Selectivity (60 dB BW/3 dB BW)at span > 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< 3Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Auto or manual Accuracy at span > 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±10%at span = 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±30%Video bandwidth (VBW)Range at span > 0 . . . . . . . . . . . . . . . .3 MHz to 3 MHz, 1-3 step, 0.003 ≤VBW/RBW ≤1Noise sidebands Offset from carrier Noise sidebands ≥1 kHz < –95 dBc/Hz ≥100 kHz < –108 dBc/Hz Figure 1-3. Noise sidebandsSpectrum Measurement Frequency offset [Hz]N o i s e s i d e b a n d [d B c /H z ]Spec Typical7Amplitude range . . . . . . . . . . . . . . . . . . . . . . . . . .displayed average noise level to +30 dBm Reference value setting range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .–100 dBm to +30 dBm Level accuracy at –20 dBm input, 50 MHz, input attenuator: 10 dB, 23 °C ±5 °C . . . . . . . . . . .< ±0.8 dB Frequency response at -20 dBm input, input attenuator: 10 dB, referenced to level at 50 MHz, 23 °C ±5 °C frequency ≥100 Hz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±1.5 dB frequency < 100 Hz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±1.3 dB Amplitude fidelity 1Log scale 2Range Amplitude fidelity (dB to reference input lever [dB][dB]0 to –30±0.05–30 to –40±0.07–40 to –50±0.15–50 to –60±0.35–60 to –70±0.8–70 to –80±1.8Linear scale 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±3%Displayed average noise level at reference value ≤–40 dBm, input attenuator: auto or 0 dB at frequency ≥1 kHz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .–120 dBm/Hz at ≥100 kHz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .–133 dBm/Hz at ≥10 MHz . . . . . . . . . . . . . . . . . . . . . . . . . . . . .(–145 + frequency/100 MHz) dBm/Hz 3Figure 1-4. Typical displayed average noise level Amplitude Characteristics1.Fidelity shows an extent of nonlinearity referenced to the reference input level.2.RBW = 10 Hz, –20 dBm ≤reference value ≤+30 dBm, reference input level = full scale input level –10 dB, 23 ±5 °C3. At start frequency ≥10 MHzNote: Refer to Input attenuator part for the definition of full scale input level.Frequency offset [Hz]A v e r a g e n o i s e l e v e l [d B m /H z ]SpecTypical8Figure 1-5. Typical on-screen dynamic range (center: 100 MHz)Spurious responses Second harmonic distortion at single tone input with full scale input level –10 dB, input signal frequency ≥100 kHz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –70 dBc, < –75 dBc (SPC)Third order inter-modulation distortion at two tones input with full scale input level –16 dB, separation ≥100 kHz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –75 dBc, < 80 dBc (SPC)Spurious at single tone input with full scale input level –10 dB, input signal frequency ≤500 MHz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< –75 dBc except for the following frequency ranges:5.6 MHz ±1 MHz, 30.6 MHz ±1 MHz, 415.3 MHz ±1 MHz Residual response at reference value setting ≤–40 dBm, input attenuator: auto or 0 dB . . . . .< –110 dBmOn-screen Dynamic Range Offset frequency [Hz]O n -s c r e e n d y n a m i c r a n g e [d B c ]9Figure 1-6. Typical dynamic range at inputs R, A, and B Input attenuator Setting range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 dB to 50 dB, 10 dB step Attenuator setting (dB) Full scale input level 0–20 dBm 10–10 dBm 200 dBm 30+10 dBm 40+20 dBm 50+30 dBm Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Auto or manual (In auto mode, the attenuator is set to 20 dB above the reference value; this ensures that the maximum signal level after the attenuator will not be greater than –20 dBm.)Input attenuator switching uncertainty at attenuator: ≤30 dB, referenced to 10 dB . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±1.0 dB at attenuator: ≥40 dB, referenced to 10 dB . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±1.5 dB Temperature drift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< ±0.05 dB/°C (SPC)Scale Log 0.1 dB/div to 20 dB/div Linear at watt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.0 x 10-12W/div at volt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.0 x 10-9V/div Measurement format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Spectrum or noise (/Hz) Display unit . . . . . . . . . . . . . . . . . . . . . . . . . . . .dBm (unit of marker: dBm, dBV, dBµV, V, W)Sweep characteristics Sweep type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Linear, list Trigger type . . . . . . . . . . . . . . . . . . . . . . . . . . .Hold, single, number of groups, continuous Trigger source . .Internal (free run), external, manual, level gate, edge gate, GPIB (bus)Sweep time (excluding each sweep setup time)RBW SPAN Typical sweep time 1 MHz 500 MHz 190 ms 100 kHz 100 MHz 300 ms 10 kHz 10 MHz 240 ms 1 kHz 1 MHz 190 ms 100 Hz 100 kHz 270 ms 10 Hz 10 kHz 2.0 s 1 Hz 1 kHz 11 s—Zero Span —1Typical Dynamic Range1.See the next item for sweep time at zero span Input level (dB)(Relative to full scale input level)D y n a m i c r a n g e (d B )Sensitivity (1 Hz RBW)Sensitivity ( 100 Hz RBW)2nd harmonic distortion 3rd order inter-modulation distortion Second Third1011Gate lengthRange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 µs to 3.2 s ResolutionRange of gate length (T I )Resolution 6 µs ≤T I ≤25 ms 0.4 µs 25 ms < T I ≤64 ms 1 µs 64 ms < T I ≤130 ms 2 µs 130 ms < T I ≤320 ms 5 µs 320 ms < T I ≤1.28 s 20 µs 1.28s < T I ≤3.2 s100 µsGate lengthRange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 µs to 3.2 sResolutionRange of gate delay (T d )Resolution 2 µs ≤T d ≤25 ms 0.4 µs 25 ms < T d ≤64 ms 1 µs 64 ms <T d ≤130 ms 2 µs 130 ms < T d ≤320 ms 5 µs 320 ms < T d ≤1.28 s 20 µs 1.28 s < T d ≤3.2 s100 µsAdditional amplitude errorLog scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< 0.3 dB (SPC)Linear scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .< 3% (SPC)Gate control modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Edge (positive/negative) or level Gate trigger input (external trigger input is used)Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTL Gate outputConnector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTLSpecifications when Option 4395A-1D6 Time-Gated Spectrum Analysis is InstalledAll specifications are identical to the standard Agilent 4395A except the following items.12Measurement functions Measurement parameters Z, Y, L, C, Q, R, X, G, B, θDisplay parameters IZI, 0z , R, X, IYI, θy , G, B, I ΓI, θγ, Γx , Γy , Cp, Cs,Lp, Ls, Rp, Rs, D, QDisplay formats•Vertical lin/log scale •Complex plane•Polar/Smith/admittance chart Sweep parameters•Linear frequency sweep•Logarithmic frequency sweep •List frequency sweep•Power sweep (in dBm unit)IF bandwidth•2,10, 30, 100, 300, 1k, 3k, 10k, 30k [Hz]Calibration•OPEN/SHORT/LOAD 3 term calibration •Fixture compensation •Port extension correction Measurement port type •7-mm Output characteristicsFrequency range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100 kHz to 500 MHz Frequency resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 MHz Output impedance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 Ωnominal Output levelwhen the measurement port is terminated by 50 Ω1 . . . . . . . . . . . . . .–56 to +9 dBm when the measurement port is open . . . . . . . . . . . . . . . . . . .0.71 mVrms to 1.26 Vrms Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0.1 dBm Level accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .±(A + B + 6 x F/(1.8 x 109))dB WhereA = 2 dBB = 0 dB (at 0 dBm ≤P ≤+ 15 dBm) or B = 1 dB (at –40 dBm ≤P < 0 dBm) or B = 2 dB (at –50 dBm ≤P < –40 dBm)F is setting frequency [Hz], P is output power settingOption 4395A-010Impedance measurementThe following specifications are applied when the 43961A impedance test kit is connected to the 4395A.1.When the measurement port is terminated with 50 Ω, the signal level at the measure-ment port is 6 dB lower than the signal level at the RF OUT port.13Measurement accuracy is specified at the connecting surface of the 7-mm connector of the Agilent 43961A under the following conditions:Warm up time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .> 30 minutes Ambient temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 °C ±5 °C,within ±1 °C from the temperature at which calibration is performedSignal level (setting) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 to +15 dBm Correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ON IFBW (for calibration and measurement) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .≤300 Hz Averaging factor (for calibration and measurement) . . . . . . . . . . . . . . . . . . . . . . . . .≥8Figure 1-7. Impedance measurement accuracyIZI - θaccuracy IZI accuracy Z a = A + (B /I Z m I + C x I Z m I) x 100 [%]θaccuracy θa = sin -1(Z a /100)Where, I Z m I is I Z I measured. A, B, and C are obtained from Figure 1-7.IYI - θaccuracy IYI accuracy Y a = A + (B x I Y m I + C /I Z m I) x 100 [%]θaccuracy θa = sin -1(Y a /100)Where, I Y m I is I Y I measured. A, B, and C are obtained from Figure 1-7.Measurement Basic Accuracy(Supplemental performancecharacteristics)Test frequency [Hz]14Display LCDSize/type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.4 inch color LCD Number of pixels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .640 x 480Effective display area . . . . . . . . . . . . . . . . . . . . . . .160 mm x 115 mm(600 x 430 dots)Number of display channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2Format single, dual (split or overwrite)Number of traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .For measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 traces For memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 traces Data math . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .gain x data – offset,gain x (data - memory) – offset,gain x (data + memory) – offset,gain x (data/memory ) – offsetData hold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Maximum hold, minimum hold MarkerNumber of markersMain marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .l for each channel Sub-marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 for each channel ∆marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 for each channel Hard copyMode . . . . . . . . . . . . . . . . . . . . . . . . . . . .Dump mode only (including color dump mode)StorageBuilt-in flexible disk driveType . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.5 inch, 1.44 MByte, or 720 KByte,1.44 MByte format is used for disk initializationMemory . . . . . . . . . . . . . . . . . . . . . . . . . . .512 KByte, can be backed up by flash memory GPIBInterface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .IEEE 488.1-1987, IEEE 488.2-1987,IEC 625, and JIS C 1901-1987 standards compatible.Interface function . . . . . . . . . . . . . . . . . . . . . . . . .SH1, AH1, T6, TEO, L4, LEO, SR1, RL1,PP0, DC1, DT1, C1, C2, C3, C4, C11, E2Data transfer formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ASCII,32 and 64 bit IEEE 754 floating point format,DOS PC format (32 bit IEEE with byte order reversed)Printer parallel portInterface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .IEEE 1284 Centronics standard compliant Printer control language . . . . . . . . . . . . . . . . . . . . . . . . . . .PCL3 printer control language Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .D-SUB (25-pin)15Common toNetwork/Spectrum/Impedance Measurement16Option 4395A-001 DC voltage/current sourceThe setting of Option 4395A-001 DC voltage/current source is independent of channel 1 and channel 2 settings.VoltageRange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .–40 V to +40 V Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 mV Current limitationat voltage setting = –25 V to +25 V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .±100 mAat voltage setting = –40 V to –25 V, 25 V to 40 V . . . . . . . . . . . . . . . . . . .±20 mA CurrentRange–20 µA to -100 mA, 20 µA to 100 mAResolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 µA Voltage limitationat current setting = –20 mA to +20 mA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .±40 V at current setting = –100 mA to –20 mA, 20 mA to 100 mA . . . . . . . . . . . . . .±25 V AccuracyVoltageat 23 °C ±5 °C . . . . . . . . . . . . . . . . . . . . . . .±(0.1% + 4 mV + I dc1[mA] x 5 [Ω] mV) Currentat 23 °C ±5 °C . . . . . . . . . . . . . . . . . . . . . . .±(0.5% + 30 µA + V dc2[V]/10 [kΩ] mA) Probe powerOutput voltage . . . . . . . . . . . . . . . . .+15 V (300 mA), –12.6 V (160 mA), GND nominal Specifications when instrument BASIC is operatedKeyboard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PS/2 style 101 English keyboard Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .mini-DIN 8 bit I/0 portConnector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .D-SUB (15-pin) Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTL Number of input/output bit . . . . . . . . . . . . . . . . . . . . . .4 bit for input, 8 bit for outputFigure 1-8. 8 bit I/O port pin assignments24-bit I/O interfaceConnector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .D-SUB (36-pin) Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTL I/O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8-bit for input or output, 16-bit for output Figure 1-9. 24-bit I/O interface pin assignment1.Current at DC source connector.2.Voltage at DC source connector.Table 1-1. Signal source assignmentPin No. Signal name Signal standard1GND0 V2INPUT1TTL level, pulse input (pulse width: 1 µs or above) 3OUTPUT1TTL level, latch output4OUTPUT2TTL level, latch output5OUTPUT PORT A0TTL level, latch output6OUTPUT PORT A1TTL level, latch output7OUTPUT PORT A2TTL level, latch output8OUTPUT PORT A3TTL level, latch output9OUTPUT PORT A4TTL level, latch output10OUTPUT PORT A5TTL level, latch output11OUTPUT PORT A6TTL level, latch output12OUTPUT PORT A7TTL level, latch output13OUTPUT PORT B0TTL level, latch output14OUTPUT PORT B1TTL level, latch output15OUTPUT PORT B2TTL level, latch output16OUTPUT PORT B3TTL level, latch output17OUTPUT PORT B4TTL level, latch output18OUTPUT PORT B5TTL level, latch output19OUTPUT PORT B6TTL level, latch output20OUTPUT PORT B7TTL level, latch output21I/O PORT C0TTL level, latch output22I/O PORT C1TTL level, latch output23I/O PORT C2TTL level, latch output24I/O PORT C3TTL level, latch output25I/O PORT D0TTL level, latch output26I/O PORT D1TTL level, latch output27I/O PORT D2TTL level, latch output28I/O PORT D3TTL level, latch output29PORT C STATUS TTL level, input mode: LOW, output mode: HIGH 30PORT D STATUS TTL level, input mode: LOW, output mode: HIGH 31WRITE STROBE SIGNAL TTL level, active low, pulse output(width: 10 µs; typical)32+5 V PULLUP33SWEEP END SIGNAL TTL level, active low, pulse output(width: 20 µs; typical)34+5 V+5 V, 100 mA MAX35PASS/FAIL SIGNAL TTL level, PASS: HIGH, FAIL: LOW, latch output36PASS/FAIL WRITE STROBE SIGNALTTL level, active low, pulse output(width: 10 µs; typical)1718Input and output characteristicsExternal reference inputFrequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 MHz ±100 Hz (SPC)Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-5 dBm to +5 dBm (SPC)Input impedance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 Ωnominal Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female Internal reference outputFrequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 MHz nominal Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 dBm (SPC)Output impedance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 Ωnominal Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female Reference oven output (Option 4395A-1D5)Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 MHz nominal Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 dBm (SPC)Output impedance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 Ωnominal Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female External trigger inputLevel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTL Pulse width (Tp) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .≥2 µs typically Polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .positive/negative selective Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female External program Run/Cont inputConnector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC female Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTL Gate output (Option 4395A-1D6)Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TTL Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC femaleFigure 1-10. Trigger signal (external trigger input)General CharacteristicsPositive trigger signalNegative trigger signal。

不同繁殖力绵羊睾丸组织新转录本注释及功能解析

第51卷第9期2023年9月西北农林科技大学学报(自然科学版)J o u r n a l o f N o r t h w e s t A&F U n i v e r s i t y(N a t .S c i .E d .)V o l .51N o .9S e p.2023网络出版时间:2023-03-08 09:41 D O I :10.13207/j .c n k i .jn w a f u .2023.09.002网络出版地址:h t t ps ://k n s .c n k i .n e t /k c m s /d e t a i l /61.1390.S .20230306.1721.002.h t m l 不同繁殖力绵羊睾丸组织新转录本注释及功能解析[收稿日期] 2022-06-07 [基金项目] 国家自然科学基金项目(32160792) [作者简介] 伏晓玉(1996-),女,甘肃秦安人,在读硕士,主要从事高原家畜繁殖生理研究㊂E -m a i l :3278467721@q q .c o m [通信作者] 王欣荣(1974-),男,甘肃临潭人,教授,硕士生导师,主要从事高原家畜繁殖生理研究㊂E -m a i l :w a n gx r @g s a u .e d u .c n 伏晓玉,柳苗苗,杨雅楠,闫尊强,王欣荣(甘肃农业大学动物科学技术学院,甘肃兰州730070)[摘要] ʌ目的ɔ完善和优化绵羊(O v i s a r i e s )基因组注释信息,解析绵羊睾丸组织在不同繁殖力水平的遗传机理㊂ʌ方法ɔ以高繁殖力绵羊品种湖羊和低繁殖力绵羊品种藏绵羊(各3只)为研究对象,采集其睾丸组织,提取R N A ,构建c D N A 文库,进行R N A -S e q 测序,对测序数据进行组装㊂利用C u f f c o m pa r e 软件对重新组装的测序数据与绵羊参考基因组进行比对分析,优化已注释基因的结构,并挖掘新转录本,对新转录本进行G O 和K E G G 通路分析㊂使用D E S e q 软件挖掘差异表达新转录本,对其进行G O 和K E G G 通路分析㊂随机选取表达显著上调和下调的新转录本各5个,采用实时荧光定量P C R (R T -qP C R )对其表达水平进行验证㊂ʌ结果ɔ对6只供试羊构建了6个独立的c D N A 文库,共获得266009566个原始读段,质量控制后得到133004783个过滤后读段;6个c D N A 文库过滤后读段的G C 含量在49.61%~51.14%,符合碱基组成规律,Q 30比例ȡ92.96%㊂对4273个已注释基因的结构进行了优化,其中5'非翻译区域(U T R )延伸基因2027个,3'U T R 延伸基因1907个,5'和3'U T R 同时延伸基因339个㊂挖掘到12178个新转录本,G O 功能分析显示,2416个新转录本注释到生物学过程㊁细胞组成和分子功能;K E G G 富集发现新转录本显著富集在单纯疱疹病毒Ⅰ型感染㊁过氧物酶体㊁河马信号通路㊂与湖羊相比,藏绵羊睾丸组织中有155个新转录本表达差异显著,其中103个表达上调,52个表达下调;G O 功能分析显示,差异新转录本富集在细胞过程㊁膜部分㊁绑定等条目;K E G G 富集发现差异新转录本显著富集在钙信号通路和核因子-κB 炎症信号通路等㊂10个差异表达新转录本的R T -q P C R 验证试验结果与R N A -S e q 结果趋势一致㊂ʌ结论ɔ从湖羊和藏绵羊睾丸组织中共挖掘出12178个新转录本,其中155个差异表达㊂这些新转录本主要通过参与细胞功能㊁生物调节和代谢途径,以及通过核因子-κB 炎症信号和钙信号通路广泛参与调控生殖细胞的增殖与分化㊁睾酮的分泌㊁免疫应答㊁C a 2+跨膜转运等途径,进而调控绵羊睾丸的发育和精子的发生㊂[关键词] 湖羊;藏绵羊;睾丸;新转录本;家畜繁殖[中图分类号] S 826.2[文献标志码] A[文章编号] 1671-9387(2023)09-0011-10A n n o t a t i o n a n d f u n c t i o n a l a n a l y s i s o f n e w t r a n s c r i pt s f r o m t e s t i c u l a r t i s s u e s o f s h e e p w i t h d i f f e r e n t f e r t i l i t yl e v e l s F U X i a o y u ,L I U M i a o m i a o ,Y A N G Y a n a n ,Y A N Z u n q i a n g ,WA N G X i n r o n g(C o l l e g e o f A n i m a l S c i e n c e a n d T e c h n o l o g y ,G a n s u A g r i c u l t u r a l U n i v e r s i t y ,L a n z h o u ,G a n s u 730070,C h i n a )A b s t r a c t :ʌO b j e c t i v e ɔT h i s s t u d y i m p r o v e d a n d o p t i m i z e d t h e a n n o t a t i o n i n f o r m a t i o n o f s h e e p ge n o m e a n d a n a l y z e d g e n e t i c m e c h a n i s m of t e s t i c u l a r t i s s u e o f s h e e p (O v i s a r i e s )a t d i f f e r e n t f e c u n d i t y l e v e l s .ʌM e t h o d ɔT h r e e H u s h e e p (h igh f e r ti l i t y )a n d t h r e e T i b e t a n s h e e p (l o w f e r t i l i t y )we r e i n c l u d e d i n t h i s s t u d y .T h e t e s t i s t i s s u e s w e r e c o l l e c t e d ,R N A w e r e e x t r a c t e d ,c D N A l i b r a r i e s w e r e c o n s t r u c t e d ,R N A -S e qs e q u e n c i n g w a s p e r f o r m e d ,a n d s e q u e n c i n g d a t a w a s a s s e m b l e d .T h e r e c o m b i n e d s e q u e n c i n g da t a w e r e c o m -p a r e d w i t h t h e s h e e p r e f e r e n c e g e n o m e u s i n g c u f f c o m p a r e s o f t w a r e t o o pt i m i z e t h e s t r u c t u r e o f a n n o t a t e d g e n e s ,m i n e n e w t r a n s c r i p t s ,a n d p e r f o r m G O a n d K E G G p a t h w a y a n a l y s i s o n t h e n e w t r a n s c r i pt s .F i v e n e w Copyright ©博看网. All Rights Reserved.t r a n s c r i p t s w i t h s i g n i f i c a n t u p-r e g u l a t i o n a n d f i v e n e w t r a n s c r i p t s w i t h d o w n-r e g u l a t e d e x p r e s s i o n w e r e r a n d o m l y s e l e c t e d a n d t h e i r e x p r e s s i o n l e v e l s w e r e v e r i f i e d b y r e a l-t i m e f l u o r e s c e n c e q u a n t i t a t i v e P C R (R T-q P C R).ʌR e s u l tɔS i x i n d e p e n d e n t c D N A l i b r a r i e s w e r e c o n s t r u c t e d,a n d a t o t a l o f266009566r a w r e a d s w e r e o b t a i n e d.A f t e r q u a l i t y c o n t r o l,133004783c l e a n r e a d s w e r e o b t a i n e d.T h e G C c o n t e n t s o f t h e s i x c D N A l i b r a r i e s w e r e49.61% 51.14%,w h i c h w a s c o n s i s t e n t w i t h t h e r u l e o f b a s e c o m p o s i t i o n,a n d t h e Q30p r o p o r t i o n s w e r eȡ92.96%.E x t e n s i o n s w e r e a c h i e v e d w i t h2027t r a n s c r i p t s f o r t h e5'u n t r a n s-l a t e d r e g i o n(U T R),1907t r a n s c r i p t s f o r t h e3'U T R r e g i o n,a n d339g e n e s f o r b o t h5'a n d3'U T R a t t h e s a m e t i m e.A t o t a l o f12178n e w t r a n s c r i p t s w e r e m i n e d a n d G O f u n c t i o n a l a n a l y s i s s h o w e d t h a t2416n e w t r a n s c r i p t s w e r e a n n o t a t e d t o b i o l o g i c a l p r o c e s s e s,c e l l c o m p o s i t i o n,a n d m o l e c u l a r f u n c t i o n s.K E G G e n r i c h-m e n t s h o w e d t h a t t h e n e w t r a n s c r i p t s w e r e s i g n i f i c a n t l y e n r i c h e d i n h e r p e s s i m p l e x v i r u s t y p eⅠi n f e c t i o n, p e r o x i s o m e,a n d h i p p o p o t a m u s s i g n a l i n g p a t h w a y s.C o m p a r e d w i t h H u s h e e p,t h e e x p r e s s i o n o f155n e w t r a n s c r i p t s i n t h e t e s t i s o f T i b e t a n s h e e p w a s s i g n i f i c a n t l y d i f f e r e n t,o f w h i c h103w e r e u p-r e g u l a t e d a n d52 w e r e d o w n-r e g u l a t e d.T h e d i f f e r e n t i a l n e w t r a n s c r i p t s w e r e e n r i c h e d i n c e l l p r o c e s s,m e m b r a n e p a r t,a n d b i n d i n g t e r m s,a c c o r d i n g t o G O f u n c t i o n a l a n a l y s i s.K E G G e n r i c h m e n t f o u n d t h a t t h e n e w t r a n s c r i p t s w e r e s i g n i f i c a n t l y e n r i c h e d i n t h e c a l c i u m s i g n a l i n g p a t h w a y a n d N F-κB i n f l a mm a t o r y s i g n a l i n g p a t h w a y.T h e R T-q P C R v a l i d a t i o n t e s t o f10d i f f e r e n t i a l l y e x p r e s s e d n e w t r a n s c r i p t s w e r e c o n s i s t e n t w i t h R N A-S e q.ʌC o n c l u s i o nɔA t o t a l o f12178n e w t r a n s c r i p t s w e r e e x t r a c t e d f r o m t h e t e s t i s t i s s u e s o f H u s h e e p a n d T i-b e t a n s h e e p,o f w h i c h155w e r e d i f f e r e n t i a l l y e x p r e s s e d.T h e s e n e w t r a n s c r i p t s r e g u l a t e d t e s t i s d e v e l o p m e n t a n d s p e r m a t o g e n e s i s b y p a r t i c i p a t i n g i n c e l l u l a r f u n c t i o n,b i o l o g i c a l r e g u l a t i o n,a n d m e t a b o l i c p a t h w a y s,a s w e l l a s c a l c i u m a n d N F-κB s i g n a l i n g p a t h w a y s i n v o l v e d i n t h e r e g u l a t i o n o f r e p r o d u c t i v e c e l l p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n,t e s t o s t e r o n e s e c r e t i o n,i mm u n e r e s p o n s e,a n d C a2+s i g n a l t r a n s d u c t i o n,s o a s t o r e g u l a t e t h e d e v e l o p m e n t o f s h e e p t e s t i s a n d s p e r m a t o g e n e s i s.K e y w o r d s:H u s h e e p;T i b e t a n s h e e p;t e s t i s;n e w t r a n s c r i p t;l i v e s t o c k b r e e d i n g藏绵羊为我国三大原始绵羊品种之一,分布于青藏高原及其毗邻的川㊁滇㊁甘等高寒地区,是当地农牧民极其重要的生产资料和经济收入来源[1]㊂藏绵羊在长期高寒低氧环境下,形成了性成熟晚㊁繁殖力低等生理特点[2]㊂湖羊是我国著名的高繁殖力绵羊品种之一,主要分布在太湖流域低海拔省份[3]㊂睾丸发育和精子发生是影响公羊繁殖力的决定性因素,作为一种高度特殊化的组织,睾丸通过产生精子和分泌雄性激素来维持绵羊的正常繁殖力㊂遗传调控是影响睾丸发育和精子发生的主要因素[4-5]㊂研究不同繁殖力绵羊的睾丸发育和精子发生的分子机制,可在绵羊育种过程中采取一些遗传调控措施,如基因突变或基因敲除等,从而提高雄性绵羊的繁殖力㊂转录组测序(R N A-S e q)是一种高通量测序技术,可为非模式动物提供丰富的基因组信息[6],也可为新转录本预测和基因结构优化提供技术支持,研究人员已利用该技术在藏猪肺[7]㊁虹鳟肝[8]㊁绵羊臂二头肌[9]等组织中挖掘出新基因和转录本㊂兰道亮等[10]应用转录组测序技术在牦牛卵巢组织中挖掘出6321个新转录本,并对7340个基因的结构进行了优化㊂目前,针对绵羊睾丸组织新转录本挖掘和基因结构优化的研究相对较少㊂为此,本研究利用R N A-S e q技术和生物信息学方法,对不同繁殖力绵羊睾丸组织新转录本进行注释和功能解析,旨在寻找与雄性绵羊繁殖能力相关的分子标记,为了解绵羊繁殖性状提供理论依据㊂1材料与方法1.1试验动物从甘肃临夏随机选取1岁健康雄性藏绵羊(低繁殖力)3只(T S1㊁T S2㊁T S3),从甘肃民勤随机选取1岁健康雄性湖羊(高繁殖力)3只(H S1㊁H S2㊁H S3),屠宰后迅速采集睾丸组织,装入冻存管置入液氮中冷冻后转至-80ħ冰箱中保存㊂1.2绵羊睾丸c D N A文库的构建及转录组测序采用T R l z o l试剂(全式金生物技术有限公司,北京)分别提取各供试羊睾丸样本的总R N A,使用A g i l e n t2100(A g i l e n t T e c h n o l o g i e s,C A,U S A)检测总R N A的完整性和浓度㊂用带有O l i g o(d T)的磁珠从提取的总R N A中富集睾丸m R N A,加入21西北农林科技大学学报(自然科学版)第51卷Copyright©博看网. All Rights Reserved.F r a g RN A m e n t a t i o n B u f f e r 将m R N A 随机打断,以打断后的m R N A 为模板,合成c D N A 链,利用AM -P u r e X P b e a d s 对c D N A 进行纯化㊂对纯化后的双链c D N A 进行末端修复,3'非翻译区域(u n t r a n s l a -t e d r e gi o n ,U R T )加A 碱基后连接测序接头,选择150~200b p 的cD N A 片段进行P C R 扩增,构建c D N A 文库㊂P C R 反应体系为:纯化后双链c D N A 模板5μL ,P C R P r i m e r M i x 2.5μL ,A m pl i f i c a t i o n M i x 12.5μL ,上游引物(序列:A G A T C G -G A A G A G C A C A C G T C T G A A C T C C A G T C A C )与下游引物(序列:A G A T C G G A A G A G C G T C G T G -T A G G G A A A G A G T G T )各1μL ,d d H 2O 3μL ㊂P C R 反应程序为:98ħ30s ;98ħ10s ,65ħ30s,72ħ30s ,14个循环;72ħ5m i n ㊂使用I l l u m i n a H i s e q T M 2500平台对构建的6个独立的c D N A 文库进行测序㊂c D N A 文库的构建和测序由生物科技公司(百迈客,青岛)完成㊂使用F a s t p v0.18.0软件对测序获得的原始读段进行过滤,去除含有接头的R e a d s 以及低质量的读段(N 的比例大于10%的读段以及质量值(Q )ɤ10的碱基占50%以上的读段),计算过滤后读段的G C 含量及Q ȡ30的碱基(Q 30)占总碱基的比例㊂使用H I S A T 2软件将过滤后读段与绵羊参考基因组(h t t p s ://w w w.n c b i .n l m.n i h .g o v )进行比对,统计比对到参考基因组上的读段占过滤后读段的比例(比对率)㊁比对到参考基因组唯一位置的读段占过滤后读段的比例(单一比对率)㊁比对到参考基因组多处位置的读段占过滤后读段的比例(多重比对率)㊁比对到参考基因组正链的读段占过滤后读段的比例(正链比对率)㊁比对到参考基因组负链的读段占过滤后读段的比例(负链比对率)㊂1.3 已注释基因结构的优化用S t r i n g T i e 软件将与绵羊参考基因组单一比对的读段进行拼接组装㊂用C u f f c o m p a r e 软件将绵羊参考基因组与组装的转录本进行比对,如果在已注释基因边界之外区域有连续的匹配读段,则将该已注释基因的非翻译区域(3'U T R 和5'U T R )向上游或下游延伸,优化已注释基因的边界[8]㊂1.4 新转录本的挖掘与功能注释将绵羊参考基因组与组装的转录本进行比对,过滤掉少于50个氨基酸残基或只包含单个外显子的编码肽链的序列,寻找未被注释的新转录本㊂对挖掘到的新转录本进行G O 功能富集分析和K E G G 信号通路富集分析㊂1.5 差异表达新转录本的挖掘使用S t r i n g Ti e 软件计算每个转录本的每百万片段中来自某一基因每千个碱基长度的数目(f r a g -m e n t s p e r k i l o b a s e o f t r a n s c r i p t s e qu e n c e p e r m i l -l i o n s m a p pe d r e a d s ,F P KM ),将F P KM 作为表达量的单位,使用D E S e q 软件对3个生物重复的新转录本表达谱进行分析,P <0.05和差异倍数(f o l dc h a n ge s ,F C )ȡ1.5的转录本被确定为差异表达转录本㊂对挖掘到的差异表达新转录本进行G O 功能富集分析和K E G G 信号通路富集分析[11]㊂1.6 差异表达新转录本的验证随机选取表达显著上调和下调的新转录本各5个,用O l i go 7.0软件设计引物,以绵羊A C T B 基因(登录号:NM _001009784.3)为内参基因,采用R T -q P C R 法验证测序结果(R N A -S e q)㊂试验所用引物由北京擎科生物科技有限公司合成,序列见表1㊂表1 本试验所用的引物信息T a b l e 1 I n f o r m a t i o n o f p r i m e r s u s e d i n t h i s s t u d y转录本/基因T r a n s c r i p /ge n e 引物序列(5'ң3')P r i m e r s e qu e n c e (5'ң3')产物长度/b pP r o d u c t s i z eN e w G e n e _26208*F :TG T C T A A A T T C A T C T G C T C C C ,R :T G A G C C T A A A T A G A C T C T G C167N e w G e n e _15127*F :G T T A C C A G C C A T C T G T G C A A G ,R :C C T A T T T G C C C T C A G A T C A C C137N e w G e n e _7273*F :G A C A A C T C C A C A C A G C C A C C T ,R :C C T G T G C G T G T T T A T C C C C T C C182N e w G e n e _14336*F :T A A A A C A A AG T G G T T A G G C A T ,R :C A C G T T A T C T A G T G A A A G G C 132N e w G e n e _16647*F :C A G A T T A A G C C C A G T G C A A ,R :T G T C T T C G G G A T C T A A C C C T T 130N e w G e n e _17517ʀF :C A C A T A TG G T G C A A A C T C T C G ,R :A A A G A C A A T A A T T C T T G C C A G T 218N e w G e n e _17659ʀF :C A G A T G T G C C G A A A T A A C T C C ,R :T A G T T G C T A A G T C A C G T C C A 249N e w G e n e _21523ʀF :TG C A C A A T G G T G T A T T A C T C G ,R :T C T G T A T A A T A G G C T C C A G T 112N e w G e n e _17837ʀF :C G T G T C C T T C A G T T C T G G T C A ,R :C A C A C T T G T T T T C C G A C G A C T 123N e w G e n e _9151ʀF :C T C C T A A T C AG T C A C G T T G ,R :T C T T T T G A A A A T T A C C A C G A A134A C T BF :G C A G A T G T G G A T C A G C A A G C ,R :T C T C G T T T T C T G C G C A A G T T133注:*表示表达显著上调,ʀ表示表达显著下调㊂N o t e :*i n d i c a t e s s i g n i f i c a n t l y u p -r e g u l a t e d e x p r e s s i o n ,ʀi n d i c a t e s s i g n i f i c a n t l y d o w n -r e g u l a t e d e x pr e s s i o n .31第9期伏晓玉,等:不同繁殖力绵羊睾丸组织新转录本注释及功能解析Copyright ©博看网. All Rights Reserved.R T -qP C R 反应体系如下:2ˑS Y B R G r e e n P C R M a s t e r M i x t u r e 10μL ,上㊁下游引物各0.8μL ,d d H 2O 6.4μL ,c D N A 2μL ㊂R T -q P C R 反应在L i g h t C y c l e r 480(R o c h e A p pl i e d S c i e n c e ,M a n n -h e i m ,G e r m a n y)上进行,其循环条件如下:95ħ30s ;95ħ5s ,60ħ30s ,68ħ1m i n ,35个循环;72ħ10s㊂使用2-ΔΔC t方法计算转录本的相对表达量[12]㊂基因表达绘图时,将相对表达量算成差异倍数以2为低的对数(l b (F C ))2 结果与分析2.1 绵羊睾丸组织c D N A 文库的构建与测序分析基于课题组先前的测序数据[13],可知共获得266009566个原始读段,质量控制后得133004783个过滤后读段,6个c D N A 文库过滤后读段的G C含量在49.61%~51.14%,符合碱基组成规律,Q 30比例ȡ92.96%(表2)㊂获得的所有原始数据均存储在N C B I 序列读取档案(S R A )中,生物项目登记号为P R J N A 841692㊂过滤后读段与绵羊参考基因组比对结果(表3)显示,6只供试羊的比对率达95.97%以上,说明匹配率较高;多重比对率为2.36%~2.57%,单一比对率为93.45%~94.99%;正链比对率为49.60%~50.33%,负链比对率为49.66%~50.34%㊂表2 藏绵羊和湖羊睾丸c D N A 文库测序结果T a b l e 2 S e q u e n c i n g o f c D N A l i b r a r i e s o f T i b e t a n s h e e p a n d H u s h e e p te s t e s c D N A 文库c D N A l i b r a r y原始读段R a w r e a d s过滤后读段C l e a n r e a d sG C 含量/%G C c o n t e n tQ 30碱基比例/%Q 30p r o po r t i o n H S 1437327622186638150.0195.09H S 2442970842214854251.1494.71H S 3441207462206037350.6093.22T S 1422993782114968950.2094.89T S 2452777642263888250.3293.08T S 3462818322314091649.6192.96表3 藏绵羊和湖羊睾丸c D N A 文库过滤后数据与参考基因组序列比对分析T a b l e 3 C o m p a r i s o n o f f i l t e r e d d a t a o f T i b e t a n s h e e p a n d H u s h e e p cD N A l i b r a r i e s w i t h r e f e r e n c e g e n o m e s e qu e n c e s %c D N A 文库c D N A l i b r a r y比对率M a p pe d r a t i o 单一比对率U n i q u e l y m a p pe d r a t i o多重比对率M u l t i p l e m a p pe d r a t i o正链比对率P o s i t i v e s t r a n d m a p pe d r a t i o 负链比对率N e g a t i v e s t r a n d m a p pe d r a t i o H S 197.4594.992.4650.3350.34H S 295.9793.612.3649.6049.66H S 396.0293.562.4649.6349.70T S 196.9394.472.4650.0550.07T S 296.0293.452.5749.7349.79T S 396.0293.492.5349.6949.732.2 绵羊睾丸组织已注释基因结构的优化表4结果显示,本研究共对4273个基因的结构进行了优化,其中5'U T R 延伸基因2027个,3'U T R 延伸基因1907个,5'U T R 和3'U T R 同时延伸基因339个;正链延伸基因2071个,负链延伸基因2202个㊂在不同染色体上随机选取一个基因,统计原位置与优化后的位置,优化结果见表5㊂表4 藏绵羊和湖羊睾丸已注释基因3'U T R 和5'U T R 延伸情况T a b l e 4 E x t e n s i o n o f 3'a n d 5'e n d s o f a n n o t a t e d g e n e s i n T i b e t a n s h e e p a n d H u s h e e p染色体I D C h r o m o s o m e I D基因数N u m b e r o fge n e s 5'U T R 延伸基因数N u m b e r o f g e n e se x t e n d e d a t5'U T R3'U T R 延伸基因数N u m b e r o f g e n e s e x t e n d e d a t 3'U T R 5'和3'U T R 同时延伸基因数N u m b e r o f g e n e s e x t e n d e d a t t h e 5'a n d 3'U T R a t t h e s a m e t i m e正链延伸基因数N u m b e r o f p o s i t i v e s t r a n d e x t e n s i o n g e n e s 负链延伸基因数N u m b e r o f n e ga t i v e s t r a n d e x t e n s i o nge n e s N C _056054.139419417129194200N C _056055.133914915634177162N C _056056.14392141873821122841西北农林科技大学学报(自然科学版)第51卷Copyright ©博看网. All Rights Reserved.表4(续) T a b l e4(c o n t i n u e d)染色体I D C h r o m o s o m e I D基因数N u m b e r o fg e n e s5'U T R延伸基因数N u m b e r o f g e n e se x t e n d e d a t5'U T R3'U T R延伸基因数N u m b e r o f g e n e se x t e n d e d a t3'U T R5'和3'U T R同时延伸基因数N u m b e r o f g e n e se x t e n d e d a t t h e5'a n d3'U T R a t t h es a m e t i m e正链延伸基因数N u m b e r o f p o s i t i v es t r a n d e x t e n s i o ng e n e s负链延伸基因数N u m b e r o f n e g a t i v es t r a n d e x t e n s i o ng e n e sN C_056057.11134655126449 N C_056058.1217115957105112 N C_056059.11236449105964 N C_056060.11867290249492 N C_056061.195434755045 N C_056062.1114644285262 N C_056063.166392432838 N C_056064.12401309317110130 N C_056065.1134695966371 N C_056066.11526178138567 N C_056067.12191069518108111 N C_056068.1145756466976 N C_056069.151261872229 N C_056070.11396264136970 N C_056071.11216050115467 N C_056072.1165481051253112 N C_056073.11083959106147 N C_056074.1123665076360 N C_056075.182433453448 N C_056076.154272162430 N C_056077.11517268117675 N C_056078.180383663842 N C_056079.155252282926 N C_056080.11607671137684其他O t h e r s844035总计T o t a l42732027190733920712202表5藏绵羊和湖羊睾丸部分已注释基因的结构优化结果T a b l e5 S t r u c t u r a l o p t i m i z a t i o n o f s o m e a n n o t a t e d g e n e s i n T i b e t a n s h e e p a n d H u s h e e p染色体编号C h r o m o s o m e I D基因G e n e延伸位置E x t e n d e d p o s i t i o n延伸链E x t e n d e d c h a i n优化前位置L o c a t i o n b e f o r eo p t i m i z a t i o n优化后位置L o c a t i o n a f t e ro p t i m i z a t i o nN C_056054.1A C A P23'U T R+193133818-193138456193133818-193140052 N C_056055.1A B HD17B3'U T R+64773032-6477432664773032-64774391 N C_056056.1A D AMT S135'U T R-2635307-26353532635307-2635473 N C_056057.1E S Y T23'U T R-121014722-121017602121014622-121017602 N C_056058.1C HA F1A5'U T R-17514550-1751465617514550-17514859 N C_056060.1B L O C1S65'U T R-63041722-6304179163041722-63042424 N C_056064.1A B I35'U T R-37090459-3709048137090459-37090775 N C_056067.1B C L2L123'U T R+55807066-5580724455807066-55807247 N C_056070.1C A B I N13'U T R+71463873-7146405271463873-71464126 N C_056078.1Z C C H C245'U T R-34240403-3424061234240403-34240626 N C_056080.1D D X3X3'U T R+40576227-4057871740576227-405787742.3绵羊睾丸组织新转录本的挖掘与功能注释本研究共挖掘出12178个新转录本㊂对挖掘到的新转录本进行G O富集分析,结果(图1)显示,有2416个新转录本注释到生物过程㊁细胞组成和分子功能㊂在生物过程中,细胞过程条目的基因数量最多;在细胞组成中,膜条目的基因数量最多;在分子功能中,绑定条目的基因数量最多㊂对挖掘到的新转录本进行K E G G通路富集分析,结果(图2)表明,新转录本显著富集在单纯疱疹病毒Ⅰ型感染㊁过氧物酶体㊁河马信号通路㊂51第9期伏晓玉,等:不同繁殖力绵羊睾丸组织新转录本注释及功能解析Copyright©博看网. All Rights Reserved.1.细胞过程;2.代谢过程;3.单组织过程;4.生物调节;5.刺激反应;6.定位化;7.信号;8.多细胞机体过程;9.细胞组成或生物发生;10.发育过程;11.多组织过程;12.免疫干细胞过程;13.运动;14.生物黏附;15.生殖过程;16.繁殖;17.增长;18.行为;19.节律过程;20.膜;21.膜部分;22.细胞;23.细胞部分;24.细胞器;25.细胞器部分;26.大分子络合物;27.膜封闭腔;28.细胞外区域;29.胞外区部分;30.突触;31.突触部分;32.其他有机体;33.其他生物体部位;34.细胞结;35.绑定;36.催化活性;37.分子传感器;38.信号传感器;39.运输;40.核酸结合转录因子活性;41.结构分子活性;42.转录因子活性,蛋白质结合;43.分子功能调节剂;44.抗氧化活性1.C e l l u l a r p r o c e s s ;2.M e t a b o l i c p r o c e s s ;3.S i n g l e -o r g a n i s m p r o c e s s ;4.B i o l o g i c a l r e g u l a t i o n ;5.R e s po n s e t o s t i m u l u s ;6.L o c a l i z a t i o n ;7.S i g n a l i n g ;8.M u l t i c e l l u l a r o r g a n i s m a l p r o c e s s ;9.C e l l u l a r c o m p o n e n t o r g a n i z a t i o n o r b i o g e n e s i s ;10.D e v e l o p m e n t a l p r o c e s s ;11.M u l t i -o r ga n i s m p r o c e s s ;12.I mm u n e s y s t e m p r o c e s s ;13.L o c o m o t i o n ;14.B i o l o g i c a l a d h e s i o n ;15.R e p r o d u c t i v e p r o c e s s ;16.R e pr o d u c t i o n ;17.G r o w t h ;18.B e h a v i o r ;19.R h y t h m i c p r o c e s s ;20.M e m b r a n e ;21.M e m b r a n e p a r t ;22.C e l l ;23.C e l l p a r t ;24.O r g a n e l l e ;25.O r ga n e l l e p a r t ;26.M a c r o m o l e c u l a r c o m p l e x ;27.M e mb r a n e -e nc l o s ed l u me n ;28.E x t r a c e l l u l a r r e g i o n ;29.E x t r a c e l l u l a r r e g i o n p a r t ;30.S y n a p s e ;31.S y n a p s e p a r t ;32.O t h e r o r g a n i s m ;33.O t h e r o r g a n i s m p a r t ;34.C e l l j u n c t i o n ;35.B i n d i n g ;36.C a t a l y t i c a c t i v i t y ;37.M o l e c u l a r t r a n s d u c e r a c t i v i t y ;38.S i gn a l t r a n s d u c e r a c t i v i t y ;39.T r a n s p o r t e r a c t i v i t y ;40.N u c l e i c a c i d b i n d i n g t r a n s c r i p t i o n f a c t o r a c t i v i t y ;41.S t r u c t u r a l m o l e c u l e a c t i v i t y;42.T r a n s c r i p t i o n f a c t o r a c t i v i t y ,p r o t e i n b i n d i n g ;43.M o l e c u l a r f u n c t i o n r e g u l a t o r ;44.A n t i o x i d a n t a c t i v i t y图1 藏绵羊和湖羊睾丸组织新转录本的G O 注释F i g .1G O a n n o t a t i o n o f a l l n e w t r a n s c r i p t s f r o m t e s t i s o f T i b e t a n s h e e p a n dH u s h e e p图2 藏绵羊和湖羊睾丸组织新转录本的K E G G 分析F i g .2 K EG G a n a l y s i s o f a l l n e w t r a n s c r i p t s f r o m t e s t i s o f T i b e t a n s h e e p a n dH u s h e e p2.4 藏绵羊和湖羊睾丸组织差异表达新转录本的挖掘与功能注释与湖羊相比,藏绵羊共发现155个差异表达的新转录本,其中103个表达上调,52个表达下调㊂对155个差异表达新转录本进行G O 功能富集分析,结果(图3)表明,在生物过程中,差异新转录本61西北农林科技大学学报(自然科学版)第51卷Copyright ©博看网. All Rights Reserved.在细胞过程的基因数量最多;在细胞组成中,差异新转录本在膜和膜部分条目的基因数量最多;在分子功能中,差异新转录本在绑定㊁催化活性条目的基因数量最多㊂对155个差异表达新转录本进行K E G G 信号通路富集分析,结果(图4)显示,大量差异表达的新转录本显著富集于核因子-κB炎症信号通路和钙信号通路等与睾丸发育和精子相关的信号通路㊂1.细胞过程;2.代谢过程;3.单组织过程;4.生物调节;5.刺激反应;6.细胞组成或生物发生;7.信号转导;8.定位化;9.多组织过程;10.膜;11.膜部分;12.细胞;13.细胞部分;14.细胞器;15.大分子络合物;16.细胞外区域;17.绑定;18.催化活性;19.结构分子活性1.C e l l u l a r p r o c e s s;2.M e t a b o l i c p r o c e s s;3.S i n g l e-o r g a n i s m p r o c e s s;4.B i o l o g i c a l r e g u l a t i o n;5.R e s p o n s e t o s t i m u l u s;6.C e l l u l a r c o m p o n e n t o r g a n i z a t i o n o r b i o g e n e s i s;7.S i g n a l i n g;8.L o c a l i z a t i o n;9.M u l t i-o r g a n i s m p r o c e s s;10.M e m b r a n e;11.M e m b r a n e p a r t;12.C e l l;13.C e l l p a r t;14.O r g a n e l l e;15.M a c r o m o l e c u l a r c o m p l e x;16.E x t r a c e l l u l a r r e g i o n;17.B i n d i n g;18.C a t a l y t i c a c t i v i t y;19.S t r u c t u r a l m o l e c u l e a c t i v i t y图3藏绵羊和湖羊睾丸组织差异表达新转录本的G O注释F i g.3G O a n n o t a t i o n o f d i f f e r e n t i a l l y e x p r e s s e d n e w t r a n s c r i p t s f r o m t e s t i s o f T i b e t a n s h e e p a n dH u s h e e p图4藏绵羊和湖羊睾丸组织差异表达新转录本的K E G G富集分析F i g.4 K EG G e n r i c h m e n t a n a l y s i s o f d i f f e r e n t i a l l y e x p r e s s e d n e w t r a n s c r i p t s f r o m t e s t i s o fT i b e t a n s h e e p a n d H u s h e e p2.5藏绵羊与湖羊睾丸组织差异表达新转录本的验证随机选取10个差异表达的新转录本进行R T-q P C R验证分析,结果(图5)显示,其表达趋势与R N A-S e q相一致,说明测序结果准确可靠㊂71第9期伏晓玉,等:不同繁殖力绵羊睾丸组织新转录本注释及功能解析Copyright©博看网. All Rights Reserved.图5 藏绵羊与湖羊睾丸差异表达新转录本的R T -qP C R 验证F i g .5 R T -q P C R v a l i d a t i o n o f d i f f e r e n t i a l l y e x p r e s s e d n e w t r a n s c r i p t s i n t e s t i s o f T i b e t a n s h e e p a n d H u s h e e p3 讨论睾丸是哺乳动物转录最复杂的器官,其结构和生理功能是雄性动物繁殖活动的基础[14-16]㊂目前,随着检测技术的改进,越来越多的与睾丸和精子发育有关的特异性m R N A 被鉴定出来[17-18]㊂R N A -S e q 技术已成为发现新转录本和基因的一种高效㊁廉价的方法㊂郭英飞等[19]利用该技术在羊布鲁氏菌中挖掘出16个新转录本;王正文等[20]利用R N A -S e q 技术在平凉红牛㊁杂交和牛(和牛ˑ平凉红牛)和西门塔尔牛3个肉牛群体中挖掘出442个新转录本,并发现有92个新转录本在3个群体中差异表达显著㊂本研究利用R N A -S e q 技术和生物信息学方法对4273个已注释绵羊基因组的基因序列信息进行了优化,挖掘出12178个新转录本,其中有2416个获得功能注释,进一步完善了绵羊转录本数据库,为绵羊基因功能研究提供了参考㊂睾丸发育是一个高度精密化的过程,主要涉及生殖细胞的增殖与分化㊁间质细胞和支持细胞的发育与成熟[21]㊂本研究发现,不同繁殖力绵羊睾丸组织差异新转录本在生物过程中的细胞过程和代谢过程条目的基因数量最多㊂睾丸由于结构原因导致其内部氧气分布不均,因此生殖细胞为了维持正常的生命活动,其能量代谢往往转向糖酵解,通过细胞水平的生物调控来实现正常生命活动所需的能量供给[22]㊂有研究发现,支持细胞产生的乳酸不仅是一种能源物质,还可以通过乳酸途径控制生精细胞的发育,以及精子细胞中蛋白质和R N A 的合成[23-25],表明糖酵解反应为绵羊生殖细胞正常生命活动提供了能量保证㊂睾丸间质细胞合成睾酮是以胆固醇为原料,间质细胞线粒体外膜上的类固醇合成急性调节蛋白促进胆固醇向线粒体内膜转运,在线粒体内膜胆固醇侧链裂解酶的催化下生成孕烯醇酮,而后在质网的羟基类固醇脱氢酶和转运蛋白的共同作用下合成睾酮[26]㊂也有研究发现,小鼠睾丸间质细胞膜上存在分子量为200k u 的黄体生成素/人绒毛膜促性腺素受体(L H /h C G 受体),该受体的数量和活性决定了睾酮分泌水平[27]㊂本研究发现,差异新转录本在细胞组成中膜部分条目的基因数量最多,由此推测这些差异新转录本可能通过调节睾丸间质细胞线粒体膜上睾酮合成酶的活性以及睾丸间质细胞膜上L H /h C G 受体的活性调节睾酮的分泌,进而调控生殖器官的发育及精子发生㊂在分子功能中,差异表达新转录本在绑定条目的基因数量最多,这可能与抑制素和激活素特异性绑定有关,抑制素可以在整个睾丸发育过程中与睾丸间质细胞特异性绑定,激活素与精子细胞上的受体或高亲和力绑定蛋白以及所有生精小管周围的位点绑定,这种绑定会在不同水平上调节生殖细胞和支持细胞的增殖或分化,并调节间质细胞的活性[28]㊂上述分析表明,抑制素和激活素这种特异性绑定也可能是引起雄性绵羊繁殖力差异的原因之一㊂炎症反应是机体调节内环境稳态的重要方式,而低氧可以作为刺激原诱发炎症反应[29-30]㊂当受低氧刺激后,细胞质中的核因子-κB 二聚体游离进入细胞核,与核内相应受体结合,调节核内基因如细胞黏附分子㊁N O 合成酶和趋化因子的转录,进而诱导各种炎性因子的过表达,进一步激活核因子-κB 炎症信号通路对外来刺激做出应答[31-32]㊂有研究发现,低氧环境会使大鼠睾丸发生炎性损伤,促进炎症反应和氧化应激反应,从而使机体产生免疫应答[33]㊂本研究中大量差异表达新转录本显著富集于核因子-κB 炎症信号通路,可能是藏绵羊在适应低氧环境的长期进化过程中,通过核因子-κB 炎症81西北农林科技大学学报(自然科学版)第51卷Copyright ©博看网. All Rights Reserved.信号通路来激活机体发生炎症反应㊂本研究差异表达新转录本显著富集在钙信号通路;F a n g等[34]研究也发现,在牦牛成熟和未成熟睾丸中钙信号通路显著富集,可见钙信号通路在睾丸发育和雄性性状维持中发挥着重要而复杂的作用㊂C a2+参与睾丸生殖细胞和体细胞的多种细胞功能,尤其是调节生殖道对内分泌激素和局部调节因子的反应[35]㊂研究发现C a2+跨细胞膜运输的改变可能会对睾丸发育和类固醇激素的合成产生影响,根据门控刺激,将钙通道分为配体依赖性钙通道(L D C C)和电压依赖性钙通道(V D C C)㊂而刺激睾丸发育的卵泡刺激素(F S H)或黄体生成素(L H)是通过睾丸支持细胞和间质细胞中的V D C C使钙离子内流[36]㊂由此推测,本研究中富集在钙信号通路上的差异表达新转录本有可能参与睾丸生殖细胞C a2+跨膜转运,进而影响睾丸的发育及精子的发生㊂[参考文献][1] X i n G S,L o n g R J,G u o X S,e t a l.B l o o d m i n e r a l s t a t u s o f g r a-z i n g T i b e t a n s h e e p i n t h e N o r t h e a s t o f t h e Q i n g h a i-T i b e t a n P l a t e a u[J].L i v e s t o c k S c i e n c e,2011,136(2/3):102-107. [2]李讨讨.基于转录组学的藏绵羊睾丸功能基因鉴定及其表达调控[D].兰州:甘肃农业大学,2021.L i T T.T r a n s c r i p t o m i c s-b a s e d i d e n t i f i c a t i o n o f f u n c t i o n a lg e n e s a n d t h e i r e x p r e s s i o n r e g u l a t i o n i n T i b e t a n s h e e p t e s t e s[D].L a n z h o u:G a n s u A g r i c u l t u r a l U n i v e r s i t y,2021.[3]罗静.湖羊睾丸发育和大小相关基因的鉴定及S N P s的筛选[D].兰州:兰州大学,2020.L u o J.I d e n t i f i c a t i o n o f g e n e s a n d S N P s r e l a t e d t o t e s t i s d e v e-l o p m e n t a n d s i z e o f H u s h e e p[D].L a n z h o u:L a n z h o u U n i v e r-s i t y,2020.[4]刘在霞,段仕,孙燕勇,等.绵羊睾丸差异基因及蛋白质互作网络关系研究[J].中国畜牧兽医,2022,49(1):1-11.L i u Z X,D u a n S,S u n Y Y,e t a l.S t u d y o n d i f f e r e n t i a l g e n e a n d p r o t e i n i n t e r a c t i o n n e t w o r k o f t e s t i s i n s h e e p[J].C h i n a A n i-m a l H u s b a n d r y&V e t e r i n a r y M e d i c i n e,2022,49(1):1-11.[5] L i B,H e X L,Z h a o Y P,e t a l.T r a n s c r i p t o m e p r o f i l i n g o f d e v e-l o p i n g t e s t e s a n d s p e r m a t o g e n e s i s i n t h e M o n g o l i a n h o r s e[J].B MC G e n e t i c s,2020,21(1):46.[6] N i e H T,Z h e n g M G,W a n g Z X,e t a l.T r a n s c r i p t o m i c a n a l y s i sp r o v i d e s i n s i g h t s i n t o c a n d i d a t e g e n e s a n d m o l e c u l a r p a t h w a y si n v o l v e d i n g r o w t h o f M a n i l a c l a m R u d i t a p e s p h i l i p p i n a r u m[J].F u n c t i o n a l&I n t e g r a t i v e G e n o m i c s,2021,21(3/4):341-352.[7]袁浩楠,杨雅楠,杨天良,等.藏猪肺组织新转录本注释及功能解析[J].农业生物技术学报,2022,30(3):485-495.Y u a n H N,Y a n g Y N,Y a n g T L,e t a l.A n n o t a t i o n s a n d f u n c-t i o n a l a n a l y s i s o f n e w t r a n s c r i p t s f r o m T i b e t a n p i g(S u s s c o f a) l u n g t i s s u e[J].J o u r n a l o f A g r i c u l t u r a l B i o t e c h n o l o g y,2022,30(3):485-495.[8]马芳,刘哲,康玉军,等.虹鳟肝组织新转录本分析及基因结构优化[J].中国实验动物学报,2019,27(2):135-142.M a F,L i u Z,K a n g Y J,e t a l.A n a l y s i s o f n o v e l t r a n s c r i p t s a n-d o p t i m i z a t i o n o f t he g e n e s t r u c t u r e i n t h e l i v e r of r a i n b o w t r o u t[J].A c t a L a b o r a t o r i n m A n i a l i s S c i e n t i a S i n i c a,2019,27(2): 135-142.[9]晁天乐.绵羊臂二头肌转录组新转录本及新长非编码R N A的鉴别与验证[D].山东泰安:山东农业大学,2015.C h a o T L.I d e n t i f i c a t i o n a n d v a l i d a t i o n o f n e w t r a n s c r i p t s a n d n e wl n c R N A s i n s h e e p b i c e p s b r a c h i i t r a n s c r i p t o m e[D].T a i a n,S h a n-d o n g:S h a n d o n g A g r i c u l t u r a l U n i ve r s i t y,2015.[10]兰道亮,熊显荣,位艳丽,等.基于R N A-S e q高通量测序技术的牦牛卵巢转录组研究:进一步完善牦牛基因结构及挖掘与繁殖相关新基因[J].中国科学:生命科学,2014,44(3):307-317.L a n D L,X i o n g X R,W e i Y L,e t a l.S t u d y o n Y a k o v a r yt r a n s c r i p t o m e b a s e d o n R N A S E Q h i g h t h r o u g h p u t s e q u e n-c i n g t e c h n o l o g y:f u r t h e r i m p r o v i n g y a k g e n e s t r u c t u r e a n dm i n i n g n e w g e n e s r e l a t e d t o r e p r o d u c t i o n[J].S c i e n t i a S i n i c a(V i t a e),2014,44(3):307-317.[11] Z h a n g L C,S u n F L,J i n H G,e t a l.A c o m p a r i s o n o f t r a n-s c r i p t o m i c p a t t e r n s m e a s u r e d i n t h e s k i n o f C h i n e s e f i n e a n dc o a r s e w o o l s h e e p b r e ed s[J].S c ie n t if i c R e p o r t s,2017,7:14301.[12] F a n L,M e n g H Y,G u o X D,e t a l.D i f f e r e n t i a l g e n e e x p r e s s i o np r o f i l e s i n p e r i p h e r a l b l o o d i n N o r t h e a s t C h i n e s e H a n p e o p l ew i t h a c u t e m y o c a r d i a l i n f a r c t i o n[J].G e n e t i c s a n d M o l e c u l a rB i o l o g y,2018,41(1):59-66.[13] F u X Y,Y a n g Y N,Y a n Z Q,e t a l.T r a n s c r i p t o m i c s t u d y o fs p e r m a t o g e n e s i s i n t h e t e s t i s o f H u s h e e p a n d T i b e t a n s h e e p[J].G e n e s,2022,13(12):2212.[14]W i t t E,B e n j a m i n S,S v e t e c N,e t a l.T e s t i s s i n g l e-c e l l R N A-s e q r e v e a l s t h e d y n a m i c s o f d e n o v o g e n e t r a n s c r i p t i o n a n dg e r m l i n e m u t a t i o n a l b i a s i n D r o s o p h i l a[J].e L i f e,2019,8:e47138.[15] B r a w a n d D,S o u m i l l o n M,N e c s u l e a A,e t a l.T h e e v o l u t i o n o fg e n e e x p r e s s i o n l e v e l s i n m a mm a l i a n o r g a n s[J].N a t u r e,2011,478(7369):343-348.[16] S o u m i l l o n M,N e c s u l e a A,W e i e r M,e t a l.C e l l u l a r s o u r c e a n dm e c h a n i s m s o f h i g h t r a n s c r i p t o m e c o m p l e x i t y i n t h e m a mm a-l i a n t e s t i s[J].C e l l R e p o r t s,2013,3(6):2179-2190. [17] Y a n g H,W a n g F,L i F Z,e t a l.C o m p r e h e n s i v e a n a l y s i s o fl o n g n o n c o d i n g R N A a n d m R N A e x p r e s s i o n p a t t e r n s i n s h e e pt e s t i c u l a r m a t u r a t i o n[J].B i o l o g y o f R e p r o d u c t i o n,2018,99(3):650-661.[18]S o n M V,T r e m o e n N H,G a u s t a d A H,e t a l.R N A s e q u e n c i n gr e v e a l s c a n d i d a t e g e n e s a n d p o l y m o r p h i s m s r e l a t e d t o s p e r mD N A i n t e g r i t y i n t e s t i s t i s s u e f r o m b o a r s[J].B M C V e t e r i n a-r y R e s e a r c h,2017,13(1):362.[19]郭英飞,王玉飞,龚春丽,等.基于R N A-S e q的羊种布鲁氏菌新转录本与非编码R N A鉴定[J].中国人兽共患病学报,91第9期伏晓玉,等:不同繁殖力绵羊睾丸组织新转录本注释及功能解析Copyright©博看网. All Rights Reserved.。

Agilent RF PNA 综合指南：RF PNA 矢量网络分析仪的配置说明书

E8356A/E8801A/N3381A 300 kHz to 3 GHz E8357A/E8802A/N3382A 300 kHz to 6 GHz E8358A/E8803A/N3383A300 kHz to 9 GHzConfiguration Guide AgilentPNA RF Network AnalyzersFull S-parameter measurements•Agilent RF PNAnetwork analyzer•Test port cables, 50 ohms •Calibration kit for applicableconnector typeThis configuration guide describes standard configuration, options, accessories,upgrade kits and compatible peripherals for RF PNA vector network analyzers.This guide should be used with the Agilent RF PNA Data Sheet for a complete description of these analyzers.System configuration summary This summary lists the main components required to form a basic measurement system. Options or peripherals may be added to provide enhanced measure-ment and data storage capability.Discontinued Product Information — For Support Reference Only —World-wide Agilent sales office contact information is available at:/find/contactus1981E8356/7/8A E8801/2/3A N3381/2/3A DescriptionAvailability Availability Availability Test Set Model#-015Configurable test set XModel#-014Configurable test set XX Power Configuration Model#-1E1Extended power range Standard X X Timebase Stability Model#-1E5Add high stability timebase Standard X X Measurement Features Model#-010Time domain capability X X X Accessories Model#-1CM Rack mount kit without handles X X X Model#-1CP Rack mount kit with handles X X X N4688A USB CD R/W drive X X X N4689AUSB HubX X X Calibration Documentation Model#-UK6Commercial calibration certificate XXXwith test dataWarranty and ServiceOne, three and five year warranty and service plans are available. Contact your local Agilent sales office for details.CalibrationCalibration is available in three or five year plans. Contact your local Agilent sales office for details.2Ordering information for RF PNA Network AnalyzerAgilent RF PNA Network Analyzer Each RF PNA instrument is an integrated network analyzer with a built-in LCD display, hard and floppy disk drives,S-parameter test set and synthesized source. The analyzer has two or three 50-ohm Type-N (f) test ports. Included with each instrument is a mouse, keyboard, CD-ROM containing a copy of online help and programming documentation,anda 1-year return-to-Agilent service warranty.❐ E8356A network analyzer,300kHzto3GHz,2port, 4receiver ❐ E8357A network analyzer,300kHzto6GHz,2port, 4receiver ❐ E8358A network analyzer,300kHzto9GHz,2port, 4receiver ❐ E8801A network analyzer,300kHzto3GHz,2port, 3receiver ❐ E8802A network analyzer,300kHzto6GHz,2port, 3receiver ❐ E8803A network analyzer,300kHzto9GHz,2port, 3receiver ❐ N3381A network analyzer,300kHzto3GHz,3port, 4receiver ❐ N3382A network analyzer,300kHzto6GHz,3port, 4receiver ❐ N3383A network analyzer,300kHzto9GHz,3port, 4receiverOptions❐ Option 010time-domain capabilityFor viewing reflection and transmission responses intime or distance domain.❐ Option 014configurable test set (except E8356/7/8A) Adds front panel access to the source, the receiversand couplers. This provides the ability to improvemeasurement sensitivity for measuring low-level signals, or to add components and other peripheral instruments for a variety of measurement applications.❐ Option 015configurable test set (E8356/7/8A only) Adds front panel access to the source output and coupler input on both ports 1 and 2. Also, 35 dB step attenuators are added between the couplers and receivers. Thiscapability provides the ability to improve measurement sensitivity for measuring low-level signals, or to addcomponents and other peripheral instruments for avariety of measurement applications.❐ Option 1CM rack mount kitAdds a rack mount (part number 5063-9216) and rail kit (E3663AC) for use without handles.❐ Option 1CP rack mount kitAdds a rack mount (part number 5063-9236) and rail kit (E3663AC) for use with previously supplied handles.❐ Option 1E170 dB step attenuator (Included as standard equipment on E8356/7/8A)Adds a built-in 70 dB step attenuator to extend theoutput power range down to -85 dBm.❐ Option 1E5high-stability time base (Included as standard equipment on E8356/7/8A)Replaces standard time base reference with a higherstability unit.DocumentationThe PNA Online Help system is available within PNA instrument in the following languages: English, German, Spanish, French, Japanese, and Chinese.The PNA Service Guide and Online Help system are available on the Web: /pna Certification options❐ Option UK6Commercial calibration certificate with test dataFor online information about Agilent’sservice and support products visit:/find/tm_services3Measurement accessoriesA complete line of RF test accessories can be found inthe Agilent RF and Microwave Test Accessories Catalog (literature number 5968-4314EN) or by visiting/find/mtaAccessories are available in these connector types: 50 ohm Type-N, 3.5 mm, 7 mm, and 7-16. Test port cables and a calibration kit should be added for a complete measurement system.A verification kit is used to verify corrected system performance.Test-port cablesTest port cables are used to connect the network analyzer to the device under test.❐ N6314A50 ohm Type-N RF cable, 300 kHz to 9 GHz Includes one 610 mm (24 in) cable with male connectors (part number 8120-8862)❐ N6315A50 ohm Type-N RF cable, 300 kHz to 9 GHz Includes one 610 mm (24 in) cable with both female and male connectors (part number 8121-0027) Calibration kitsMechanical calibration kits include standards, such as opens, shorts and loads, which are measured by the network analyzer for increased measurement accuracy. Electronic calibration (ECal) kits replace mechanical calibration standards with one solid-state calibration module that is controlled by the network analyzer to present many different impedances to the test ports. Afull two-port calibration can be performed quickly with a single connection.This technique reduces operator errors and connector wear and abrasion.Choose a calibration kit for each connector type to be used.Economy, includes:• open standards (male and female)• short standards (male and female)• fixed-termination standards (male and female)• in-series adaptersStandard, includes the devices in the economy kit and adds:• connector toolsPrecision, includes the devices in the economy kit and adds:• 50 ohm airline for TRL calibration• TRL adapters• connector tools For devices with Type-N connectors Mechanical calibration kits❐ 85032F economy: 30 kHz to 9 GHz. Includes:85032-60017 Type-N (m) fixed load85032-60018 Type-N (f) fixed load85032-60013 Type-N (m) open85032-60014 Type-N (f) open85032-60016 Type-N (m) short85032-60015 Type-N (f) short❐ Option 100adds:85032-60021 Type-N (f) to Type-N (f) adapter❐ Option 200adds:85032-60019 Type-N (m) to Type-N (m) adapter❐ Option 300adds:85032-60020 Type-N (m) to Type-N (f) adapter❐ Option 500adds:85054-60001 Type-N (f) to 7 mm adapter (two included) 85054-60009 Type-N (m) to 7 mm adapter (two included)❐ 85054D economy: 45 MHz to 18 GHz. Includes: 85054-60025 Type-N (m) short85054-60026 Type-N (f) short85054-60027 Type-N (m) open85054-60028 Type-N (f) open85054-60031 Type-N (f) to 7 mm adapter85054-60032 Type-N (m) to 7 mm adapter85054-60037 Type-N (f) to Type-N (f) adapter85054-60038 Type-N (m) to Type-N (m) adapter85054-60046 Type-N (m) fixed load85054-60047 Type-N (f) fixed loadElectronic calibration kits❐ 85092C RF ECal: 300 kHz to 9 GHz, 2 ports. Includes:❐ Option MOF module with:85092-60008 Type-N (f) to Type-N (m) RF ECal module ❐ Option 00M module with:85092-60009 Type-N (m) to Type-N (m) RF ECal module ❐ Option 00F module with:85092-60010 Type-N (f) to Type-N (f) RF ECal module ❐ Option 00A adds:85054-60037 Type-N (f) to Type-N (f) adapter85054-60038 Type-N (m) to Type-N (m) adapter4For devices with 3.5 mm or SMA connectors (see Adapters section for information about the Agilent 11878A 3.5 mm adapter kit)Mechanical calibration kits❐ 85033E economy: 30 kHz to 9 GHz. Includes:85033-60016 3.5 mm (m) load85033-60017 3.5 mm (f) load85033-60018 3.5 mm (m) open85033-60019 3.5 mm (f) open85033-60020 3.5 mm (m) short85033-60021 3.5 mm (f) short8710-1761 torque wrench❐ Option 100adds:85027-60005 3.5 mm (f) to 3.5 mm (f) adapter❐ Option 200adds:85027-60007 3.5 mm (m) to 3.5 mm (m) adapter❐ Option 300 adds:85027-60006 3.5 mm (m) to 3.5 mm (f) adapter❐ Option 400adds:1250-1744 3.5 mm (f) to Type- N 50 ohm (m) adapter1250-1743 3.5 mm (m) to Type- N 50 ohm (m) adapter 1250-1745 3.5 mm (f) to Type- N 50 ohm (f) adapter1250-1750 3.5 mm (m) to Type- N 50 ohm (f) adapter ❐ Option 500adds:1250-1746 3.5 mm (m) to 7 mm adapter (two included) 1250-1747 3.5 mm (f) to 7 mm adapter (two included)❐ 85052C precision TRL: 45 MHz to 26.5 GHz. Includes: 00902-60003 3.5 mm (m) fixed load00902-60004 3.5 mm (f) fixed load85052-60006 3.5 mm (m) short85052-60007 3.5 mm (f) short85052-60008 3.5 mm (m) open85052-60009 3.5 mm (f) open85052-60032 3.5 mm (f) to 3.5 mm (f) adapter85052-60033 3.5 mm (m) to 3.5 mm (m) adapter85052-60034 3.5 mm (f) to 3.5 mm (m) adapter85052-60035 3.5 mm short TRL line85052-60036 3.5 mm long TRL line❐ 85052D economy: 45 MHz to 26.5 GHz. Includes: 00902-60003 3.5 mm (m) fixed load00902-60004 3.5 mm (f) fixed load85052-60006 3.5 mm (m) short85052-60007 3.5 mm (f) short85052-60008 3.5 mm (m) open85052-60009 3.5 mm (f) open85052-60012 3.5 mm (f) to 3.5 mm (f) adapter85052-60013 3.5 mm (f) to 3.5 mm (m) adapter85052-60014 3.5 mm (m) to 3.5 mm (m) adapter Electronic calibration kits❐ 85093C RF ECal: 300 kHz to 9 GHz, 2 ports. Includes: 85093-60008 3.5 mm (f) to 3.5 mm (m) RF ECal module ❐ Option 00M module with:85093-60009 3.5 mm (m) to 3.5 mm (m) RF ECal module ❐ Option 00F module with:85093-60010 3.5 mm (f) to 3.5 mm (f) RF ECal module ❐ Option 00A adds:85052-60012 3.5 mm (f) to 3.5 mm (f) adapter85052-60014 3.5 mm (m) to 3.5 mm (m) adapter For devices with 7 mm connectors(see A dapters section for information about Agilent 7 mm adapters)Mechanical calibration kits❐ 85031B economy: 30 kHz to 6 GHz. Includes:00909-60008 7 mm coax termination85031-60001 7 mm open/short❐ 85050C precision TRL: 45 MHz to 18 GHz. Includes: 00909-60008 7 mm coax termination85050-60003 7 mm to 7 mm airline85050-60005 7 mm to 7 mm TRL adapter85050-60006 7 mm fixed broadband load85050-80008 7 mm short85050-80009 7 mm short collet85050-80010 7 mm open❐ 85050D economy: 45 MHz to 18 GHz. Includes: 85050-60006 7 mm fixed broadband load85050-80007 7 mm short85050-80010 7 mm openElectronic calibration kits❐ 85091C RF ECal: 300 kHz to 9 GHz, 2 ports. Includes: 85091-60004 7 mm to 7 mm RF ECal module5For devices with 7-16 connectors(see Adapters section for information about the Agilent 11906B 7-16 to Type-N adapter kit)Mechanical calibration kits❐ 85038A standard: 30 kHz to 7.5 GHz. Includes: 85038-80002 7-16 (f) open85038-80003 7-16 (m) open85038-80004 7-16 (f) short85038-80005 7-16 (m) short85038-80006 7-16 (f) fixed load85038-80007 7-16 (m) fixed load8710-2175 torque wrench8710-2174 open-end wrench❐ 85038F economy: 30 kHz to 7.5 GHz. Includes: 85038-80002 7-16 (f) open85038-80004 7-16 (f) short85038-80006 7-16 (f) fixed load11906-80016 7-16 (f) to 7-16 (f) adapter❐ 85038M economy: 30 kHz to 7.5 GHz. Includes: 85038-80003 7-16 (m) open85038-80005 7-16 (m) short85038-80007 7-16 (m) fixed load11906-80015 7-16 (m) to 7-16 (m) adapter Electronic calibration kits❐ 85098C RF ECal: 300 kHz to 7.5 GHz, 2 ports. Includes:❐ Option MOF module with:85098-60007 7-16 (m) to 7-16 (f) RF ECal module ❐ Option 00F module with:85098-60009 7-16 (f) to 7-16 (f) RF ECal module❐ Option 00M module with:85098-60008 7-16 (m) to 7-16 (m) RF ECal module ❐ Option 00A adds:11906-80015 7-16 (m) to 7-16 (m) adapter11906-80016 7-16 (f) to 7-16 (f) adapter Verification kitsAll Agilent Technologies verification kits include:• precision Z°airline• mismatched airline• fixed attenuators• traceable measured data and uncertainties❐ 85055A300 kHz to 18 GHz Type-N kitIncludes attenuators, airlines and mismatch airline with data on a 3.5 inch disk for use in confirming accuracy enhanced system measurement performance, traceable to national standards. Test procedure is provided in the service manual.❐ 85053B300 kHz to 26.5 GHz 3.5 mm kitIncludes attenuators, airlines and mismatch airline with data on a 3.5 inch disk for use in confirming accuracy enhanced system measurement performance, traceable to national standards. Test procedure is provided in the service manual.6Adapters❐ 11853A 50 ohm Type-N accessory kit. Includes:1250-1472 Type-N (f) to Type-N (f) adapter (two included)1250-1475 Type-N (m) to Type-N (m) adapter (two included)11511A Type-N (f) short11512A Type-N (m) short❐ 11878A Type-N to 3.5 mm adapter kit. Includes:1250-1744 3.5 mm (f) to Type-N 50 ohm (m) adapter1250-1743 3.5 mm (m) to Type-N 50 ohm (m) adapter1250-1745 3.5 mm (f) to Type-N 50 ohm (f) adapter1250-1750 3.5 mm (m) to Type-N 50 ohm (f) adapter❐ 11524A7 mm to Type-N (f) adapter❐ 11525A7 mm to Type-N (m) adapter❐ 11906A7-16 to 7-16. Includes:7-16 (m) to 7-16 (m) adapter7-16 (f) to 7-16 (f) adapter7-16 (m) to 7-16 (f) adapter (two included)❐ 11906B7-16 to Type-N. Includes:Type-N (m) to 7-16 (m) adapterType-N (f) to 7-16 (f) adapterType-N (f) to 7-16 (m) adapterType-N (m) to 7-16 (f) adapter❐ 11854A50 ohm BNC accessory kit. Includes:1250-0929 BNC (m) short1250-1473 BNC (m) to Type-N (m) adapter (two included)1250-1474 BNC (f) to Type-N (f) adapter (two included)1250-1476 BNC (f) to Type-N (m) adapter (two included)1250-1477 BNC (m) to Type-N (f) adapter (two included)7General accessoriesUSB❐N4688A CD read/write driveProvides an external read/write CD drive with aUSB cable.❐N4489 USB HubProvides an external USB hub with at least 4-ports and a USB cable.Probe❐85024A high-frequency probeProvides high-impedance in-circuit test capability from 300 kHz to 3 GHz.Power meters and sensorsRecommended for self support, adjustments and performance tests to verify proper instrument operation.❐ E4418B single-channel power meter❐ E4419B dual-channel power meter❐ 8482A power sensor, 100 kHz to 4.2 GHz, Type-N (m), 100 mW❐ E4412A CW power sensor, 10 MHz to 18 GHz, Type-N (m), 200 mW Amplifiers1❐ 8347A RF power amplifier, 100 kHz to 3 GHz, 25 dB gain, power out: +20 dBm❐ 83006A power amplifier, 10 MHz to 26.5 GHz, 20 dB gain, power out: +18dBm to 10 GHz or +16 dBm to 20 GHz or +14 dBm to 26.5 GHz❐ 83017A power amplifier, 50 MHz to 26.5 GHz, 25 dB gain, power out: +20 dBm to 20 GHz, or +15 dBm to 26.5 GHz ❐ 83018A power amplifier, 2 to 26.5 GHz, 27 dB gain to20 GHz or 23 dB to 26.5 GHz, power out: +24 dBm to20 GHz or +21 dBm to 26.5 GHz❐ 83020A power amplifier, 2 to 26.5 GHz, 30 dB gain to20 GHz or 27 dB to 26.5 GHz, power out: +30 dBm to20 GHz or +26 dBm to 26.5 GHzCouplers❐ 87300B coaxial coupler, 1 to 20 GHz, SMA (f), 10 dB coupling❐ 87300C coaxial coupler, 1 to 26.5 GHz, 3.5 mm (f), 10 dB couplingEquipment racks and case❐ 5063-9223rack mount flange kit, for use with handles;includes handles2❐ 5063-9216rack mount kit, for use without handles; may be ordered as option 1CM❐ 5063-9236rack mount kit, for use with previously supplied handles; may be ordered as option 1CP❐ E3663AC rail kit, included with option 1CM and 1CP.❐ 9211-2658transit case1. RF connectors: 3.5 mm (f) on RF input and output; BNC (f) detector out.Type-N (f) on RF input and output for 8347A2. A PNA Series analyzer is supplied with handles.8ApplicationsMaterial measurement❐85070D High-temperature dielectric probe kitThe 85070D allow the measurement of the dielectricproperties of materials quickly and conveniently.Measurements made with this probe are nondestructiveand require no sample preparation. The dielectric probeis well suited for measurements of liquid, semisolid andflat solid materials. Measurement results can be viewedin a variety of formats (εr', εr ", tan δor Cole-Cole).The supplied software can be run in the PNA analyzeror on a PC.❐85071D Materials measurement softwareThe material software calculates the permittivity andpermeability of material samples placed in a coaxialairline or a rectangular waveguide. The measurementtechnique works well for solid materials that can bemachined to fit precisely inside a transmission line.Measurement results can be viewed in a variety offormats (εr', εr ", tan δ, µr', µr ", tan δm or Cole-Cole).The software can be run in the PNA analyzer or on a PC.Filter tuning❐N4261A Filter tuning softwareThis software will significantly reduce filter tuningtraining times and increase manufacturing throughput.Through the graphical user interface, visual trainingindicators show when the individual resonators andcouplers in a filter have been tuned within specifications.The indicators also show the user when and how to tuneeach filter element, reducing the number of times eachelement has to be re-tuned before the filter meetsspecifications.9PeripheralsThe following peripherals may be used with all RF PNAs. Other peripherals not listed here may also be compatible with these instruments.Monitors❐ VGA-compatible monitorPrinters❐ USB, LAN, parallel or serial printers withMicrosoft®Windows®2000 printer driver Interface cablesChoose the appropriate cables to connect each peripheral to the network analyzer.❐ 10833A GPIB cable, 1.0 m (3.3 ft)❐ 10833B GPIB cable, 2.0 m (6.6 ft)❐ 10833D GPIB cable, 0.5 m (1.6 ft)Upgrade kitsUpgrade kits for the E8356/7/8A, E8801/2/3A, and N3381/2/3AUpgrade kits are available to add options after initialpurchase.To order an upgrade kit for a PNA, order theanalyzer’s model number followed by a “U”, then indicate the option to be added:PNA Series RF Network Analyzer Upgrade KitsE8356/7/8AU 2-port, 4 receiver, 300kHz – 3/6/9GHz Upgrade Kits E8801/2/3AU 2-port, 3 receiver, 300kHz – 3/6/9GHz Upgrade Kits N3381/2/3AU 3-port, 4 receiver, 300kHz – 3/6/9GHz Upgrade KitsOptionsTo add options to a product, use the following ordering scheme Model:Model#U (ex: E8358AU)Model Options:Model#U-opt# (ex: E8356AU-015)❐ Option 006 for E8356A, E8801A or N3381A only.Adds 6 GHz operation. Includes installation at an Agilentservice center. Instrument calibration is required for anadditional fee.❐ Option 009 for E8356A, E8357A E8801A, E8802A, N3381A or N3382A only.Adds 9 GHz operation. Includes installation at an Agilentservice center. Instrument calibration is required for anadditional fee.❐ Option 010time-domain upgrade kit (part number E8356-60101) The serial number of the PNA to be retrofitted must bespecified when ordering this kit. Installation is notincluded.❐ Option 014for E8801/2/3A and N3381/2/3A only.Configurable test set upgrade kit (part number E8801-60103or N3381-60101, depending on model number). Includesinstallation at an Agilent service center.❐ Option 015for E8356A, E8357A or E8358A only.Configurable test set upgrade kit (part number E8356-60102).Includes installation at an Agilent service center.❐ Option 098 CPU board upgrade for E8356/7/8A built before November, 2001 (part number E8356-60103). Includesinstallation at an Agilent service center.❐ Option 099firmware upgrade (part number E8356-60103) Provides the latest revision of firmware for thePNA Series on CD-ROM. Firmware is user-installable.Installation requires USB CD-ROM drive or externalcomputer connected via LAN. The latest firmware isalso available from our web site. Visit our web page at/find/pna❐ Option 1E1 for E8801/2/3A and N3381/2/3A only.Adds a built-in step attenuator to extend the output powerrange down to -85 dBm (part number E8801-60104).Includes installation at an Agilent service center.❐ Option 1E5 for E8801/2/3A and N3381/2/3A only.Replaces standard frequency reference with a higherstability unit (part number E8801-60105). Includesinstallation at an Agilent service center.Contact your local Agilent sales or service office forfurther information.1011Literature and informationPNA RF and Microwave Brochure literature number 5968-8472E RF PNA Data Sheetliterature number 5980-1236EKey web resourcesVisit the PNA network analyzer home page at:/find/pnaFor additional application information and to download application notes, visit:/find/appcentral/find/emailupdatesGet the latest information on the products and applications you select.Microsoft ® and Windows ® are U.S. registered trademarks of Microsoft Corporation.Agilent T echnologies’ T est and Measurement Support, Services, and Assistance Agilent T echnologies aims to maximize the value you receive, while minimizing your risk and problems. We strive to ensure that you get the test and measure-ment capabilities you paid for and obtain the support you need. Our extensive support resources and services can help you choose the right Agilent products for your applications and apply them successfully. Every instrument and system we sell has a global warranty. T wo concepts underlie Agilent’s overall support policy: “Our Promise” and “Your Advantage.”Our PromiseOur Promise means your Agilent test and measurement equipment will meet its advertised performance and functionality. When you are choosing new equipment, we will help you with product information, including realistic perfor-mance specifications and practical recommendations from experienced test engineers. When you receive your new Agilent equipment, we can help verify that it works properly and help with initial product operation.Your AdvantageYour Advantage means that Agilent offers a wide range of additional expert test and measurement services, which you can purchase according to your unique technical and business needs. Solve problems efficiently and gain a competitive edge by contracting with us for calibration, extra-cost upgrades, out-of-warranty repairs, and onsite education and training, as well as design, system integration,project management, and other professional engineering services. Experienced Agilent engineers and technicians worldwide can help you maximize your produc-tivity, optimize the return on investment of your Agilent instruments and systems,and obtain dependable measurement accuracy for the life of those products.United States:Korea:(tel) 800 829 4444(tel) (080) 769 0800(fax) 800 829 4433(fax) (080) 769 0900Canada:Latin America:(tel) 877 894 4414(tel) (305) 269 7500(fax) 800 746 4866Taiwan :China:(tel) 0800 047 866(tel) 800 810 0189(fax) 0800 286 331(fax) 800 820 2816Other Asia Pacific Europe:Countries:(tel) 31 20 547 2111(tel) (65) 6375 8100Japan:(fax) (65) 6755 0042(tel) (81) 426 56 7832Email:*****************(fax) (81) 426 56 7840Contacts revised: 09/26/05For more information on Agilent Technologies’ products, applications or services,please contact your local Agilent office. The complete list is available at:/find/contactusProduct specifications and descriptions in this document subject to change without notice.© Agilent Technologies, Inc. 2003, 2004, 2006Printed in USA, July 13, 20065980-1235E/find/emailupdatesGet the latest information on the products and applications you select.Agilent Email Updates/find/agilentdirectQuickly choose and use your test equipment solutions with confidence.Agilent DirectAgilent Open/find/openAgilent Open simplifies the process of connecting and programming test systems to help engineers design, validate and manufacture electronic products. Agilent offers open connectivity for a broad range of system-ready instruments, open industry software, PC-standard I/O and global support, which are combined to more easily integrate test system development.。

大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析

中国畜牧兽医 2022,49(8):2855-2868C h i n aA n i m a lH u sb a n d r y &V e t e r i n a r y Me d i c i ne 大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析聂靖茹1,2,马黎3,严达伟1,邓俊4,张浩2,张博2,刘金桥1,董新星1(1.云南农业大学动物科学技术学院,昆明650201;2.中国农业大学动物科学技术学院,北京100193;3.云南农业职业技术学院畜牧兽医学院,昆明650212;4.云南省畜牧总站,昆明650224)摘要:ʌ目的ɔ筛选大型迪庆藏猪不同生长阶段肌内脂肪含量差异的关键基因并分析其调控途径㊂ʌ方法ɔ选择胎次相同㊁出生日期及体重相近的大型迪庆藏猪36头,随机分为3组,在相同条件下进行育肥试验,分别在体重达40㊁80及120k g 左右时屠宰,每组采集3头猪的背最长肌,采用R N A -S e q 技术进行转录组测序,测序数据进行拼接㊁比对和注释,筛选与脂肪沉积相关的差异显著基因进行功能富集㊁S T E M 分析,构建基因互作网络,并进行实时荧光定量P C R 验证㊂ʌ结果ɔ40k g v s 80k g ㊁80k g v s 120k g 与40k g v s 120k g 阶段分别检测到730㊁981及735个基因差异显著表达;S T E M 分析共有4个差异显著模块,模块11㊁14的基因集先显著上调后轻微下调;模块9㊁10的基因集先显著上调后显著下调㊂差异基因互作网络显示,40k g v s 80k g 阶段,E G R 1㊁E G R 2㊁P R K A G 2㊁N O R -1和A T F 3基因位于网络核心,E G R 1和E G R 2基因表达下调,P R K A G 2㊁N O R -1和A T F 3基因表达上调;80k g v s 120k g 阶段,F O X O 1㊁P D K 4㊁P P A R D ㊁P P A RG C -1㊁L I P E ㊁A T F 3和S T A T 1基因位于网络核心,F O X O 1㊁P P A R D ㊁P P A R G C -1基因表达下调,P P A R G 基因通过级联调控引起S T A T 1基因表达下调,L I P E 和A T F 3基因表达上调;40k g v s 120k g 阶段,A T F 3㊁N O R -1㊁E G R 1㊁E G R 2和S T A T 1基因位于网络核心,E G R 1㊁E G R 2和S T A T 1基因表达下调,A T F 3和N O R -1基因表达上调㊂A T F 3㊁F O X O 1等10个基因的实时荧光定量P C R 验证结果与转录组测序结果一致㊂ʌ结论ɔE G R 1㊁F O X O 1等基因作为核心基因参与大型迪庆藏猪肌内脂肪调控,不同生长阶段参与调控的核心基因并不完全相同,结果可丰富中国地方猪肌内脂肪调控基础数据,为大型迪庆藏猪肌内脂肪含量的遗传改良提供参考㊂关键词:大型迪庆藏猪;R N A -S e q;肌内脂肪沉积;差异表达基因;调控途径中图分类号:S 828文献标识码:AD o i :10.16431/j .c n k i .1671-7236.2022.08.002 开放科学(资源服务)标识码(O S I D ):收稿日期:2022-02-09基金项目:云南省乡村振兴科技专项(202104B I 090021);云南省高校科技创新团队支持计划(云南省高校高原山地畜禽抗逆性基因发掘与利用科技创新团队)联系方式:聂靖茹,E -m a i l :136********@163.c o m ㊂通信作者马黎,E -m a i l :935936939@q q .c o m ;董新星,E -m a i l :86127447@q q.c o m A n a l y s i s o fD i f f e r e n t i a l l y E x p r e s s e dG e n e s a n dR e g u l a t i o nP a t h w a ys o f I n t r a m u s c u l a r F a tD e p o s i t i o n i nL a r g eD i q i n g T i b e t a nP ig s a tD i f f e r e n tG r o w t hS t a ge s N I EJ i n gr u 1,2,MA L i 3,Y A N D a w e i 1,D E N GJ u n 4,Z H A N G H a o 2,Z H A N GB o 2,L I UJ i n q i a o 1,D O N G X i n x i n g1(1.C o l l e g e o f A n i m a lS c i e n c e a n dT e c h n o l o g y ,Y u n n a nA g r i c u l t u r a lU n i v e r s i t y ,K u n m i n g650201,C h i n a ;2.C o l l e g e o f A n i m a lS c i e n c e a n dT e c h n o l o g y ,C h i n aA g r i c u l t u r a lU n i v e r s i t y ,B e i j i n g 100193,C h i n a ;3.D e p a r t m e n t o f A n i m a lH u s b a n d a r y a n dV e t e r i n a r y Me d i c i n e ,Y u n n a nV o c a t i o n a l a n dT e c h n i c a lC o l l e g e of Ag r i c u l t u r e ,K u n m i n g 650212,C h i n a ;4.Y u n n a nA n i m a lH u s b a n d r y S t a t i o n ,K u n m i n g 650224,C h i n a )A b s t r a c t :ʌO b j e c t i v e ɔT h i ss t u d y w a sa i m e d t o s c r e e n t h e k e y ge n e s o ft h e d if f e r e n c e o f中国畜牧兽医49卷i n t r a m u s c u l a r f a t(I M F)c o n t e n to f l a r g eD i q i n g T i b e t a n p i g s(T P s)a td i f f e r e n t g r o w t hs t a g e s a n d a n a l y z e i t s r e g u l a t i o n p a t h w a y.ʌM e t h o dɔT h e r ew e r e t h i r t y-s i xT P sw i t ht h es a m e p a r i t y, d a t e o f b i r t ha n dw e i g h tw e r e r a n d o m l y d i v i d e d i n t o t h r e e g r o u p s.T h e f a t t e n i n g t e s tw a s c a r r i e d o u t u n d e rt h es a m ec o n d i t i o n s.T h e y w e r es l a u g h t e r e d w h e nt h e w e i g h tw a sa b o u t40,80a n d 120k g,r e s p e c t i v e l y.T h el o n g i s s i m u s d o r s i m u s c l e(L D)o ft h r e e p i g si n e a c h g r o u p w a s c o l l e c t e d,a n dt h et r a n s c r i p t o m ew a ss e q u e n c e db y R N A-S e q.T h es e q u e n c i n g d a t aw a ss p l i c e d,c o m p a r ed a n da n n o t a te d,t h es i g n if i c a n t l y d i f f e r e n t i a l l y e x p r e s s e dg e n e s(D E G s)r e l a t e dt oI M Fd e p o s i t i o n w e r e s c r e e n e d,a n d t h e g e n ei n t e r a c t i o n n e t w o r k w a s c o n s t r u c t e d b y f u n c t i o n a le n r i c h m e n ta n d S T E M a n a l y s i s.T h e r e s u l t s w e r e v e r if i e d b y R e a l-t i m e q u a n t i t a t i v e P C R.ʌR e s u l tɔT h e r ew e r e730,981a n d735g e n e sw e r es i g n i f i c a n t l y d i f f e r e n t i a l l y e x p r e s s e d i n40k g v s 80k g,80k g v s120k g a n d40k g v s120k g s t a g e s,r e s p e c t i v e l y.Th e r e w e r ef o u r m o d u l e s wi t h s i g n i f i c a n t d i f f e r e n c e s i nS T E M a n a l y s i s.T h e g e n es e t so fm o d u l e s11a n d14w e r es i g n i f i c a n t l y u p-r e g u l a t e d a n d t h e n s l i g h t l y d o w n-r e g u l a t e d.T h e g e n e s e t s o f m o d u l e s9a n d10w e r e s i g n i f i c a n t l y u p-r e g u l a t e d a n dt h e n d o w n-r e g u l a t e d.T h e d i f f e r e n t i a l g e n ei n t e r a c t i o n n e t w o r k s h o w e d t h a ta t40k g v s80k g s t a g e,E G R1,E G R2,P R K A G2,N O R-1a n d A T F3g e n e s w e r e l o c a t e d i n t h e c o r e o f t h e n e t w o r k,E G R1a n d E G R2g e n e sw e r e d o w n-r e g u l a t e d,P R K A G2,N O R-1a n d A T F3g e n e sw e r e u p-r e g u l a t e d.A t80k g v s120k g s t a g e,F O X O1,P D K4,P P A R D,P P A R G C-1, L I P E,A T F3a n d S T A T1g e n e sw e r e l o c a t e d i nt h ec o r eo f t h en e t w o r k,F O X O1,P P A R D a n d P P A R G C-1g e n e sw e r ed o w n-r e g u l a t e d,a n d P P A R G g e n ec a u s e d S T A T1g e n ed o w n-r e g u l a t i o n t h r o u g hc a s c a d e r e g u l a t i o n,L I P E a n d A T F3g e n e sw e r eu p-r e g u l a t e d.A t40k g v s120k g s t a g e, A T F3,N O R-1,E G R1,E G R2a n d S T A T1g e n e sw e r e l o c a t e d i nt h e c o r eo f t h en e t w o r k,E G R1, E G R2a n d S T A T1g e n e sw e r e d o w n-r e g u l a t e d,A T F3a n d N O R-1g e n e sw e r eu p-r e g u l a t e d.R e a l-t i m e q u a n t i t a t i v eP C Rr e s u l t s o f t e n g e n e s s u c h a s A T F3a n d F O X O1w e r e c o n s i s t e n tw i t hR N A-S e q r e s u l t s.ʌC o n c l u s i o nɔT h i ss t u d y s c r e e n e ds o m e g e n e s l i k e E G R1a n d F O X O1p a r t i c i p a t e i n t h e r e g u l a t i o no f I M F i nT P s a s c o r e g e n e s,w h i c h i n v o l v e d i n r e g u l a t i o na t d i f f e r e n t s t a g e sw e r e n o t e x a c t l y t h es a m e.T h er e s u l t sc o u l de n r i c ht h eb a s i cd a t ao f I M Fr e g u l a t i o n i nl o c a l p i g s i n C h i n a,a n d p r o v i d e a r e f e r e n c e f o r t h e g e n e t i c i m p r o v e m e n t o f I M Fc o n t e n t i nT P s.K e y w o r d s:l a r g eD i q i n g T i b e t a n p i g s;R N A-S e q;i n t r a m u s c u l a r f a td e p o s i t i o n;d i f f e r e n t i a l l y e x p r e s s e d g e n e;r e g u l a t i o n p a t h w a y肌内脂肪(i n t r a m u s c u l a r f a t,I M F)含量是肉质评定的首选指标,影响猪肉的风味㊁嫩度和多汁性[1],筛选调控猪肌内脂肪沉积的功能基因并解析其调控机制,对猪肉品质的遗传改良具有重要意义㊂随着高通量测序技术的发展,一些影响猪脂肪沉积的功能基因和转录调控因子被发现㊂李明洲等[2]研究发现,太湖猪在不同月龄脂肪细胞体积㊁肌内脂肪含量均高于长白猪,且在3月龄后差距逐渐明显; W o o d等[3]研究发现,维生素D受体(v i t a m i n D r e c e p t o r,V D R)通过抑制C C A A T/增强子结合蛋白α(C C A A T/e n h a n c e r b i n d i n g p r o t e i n s a l p h a,C/ E B Pα)和过氧化物酶体增殖物激活受体γ(p e r o x i s o m e p r o l i f e r a t o r s-a c t i v a t e dr e c e p t o r g a m m a,P P A Rγ)抑制脂肪生成;T a n g等[4]研究发现,转录因子S p1基因是前脂肪细胞向脂肪细胞分化所必需的关键基因;H u a n g等[5]研究发现,X L O C_046142㊁X L O C_ 004398和X L O C_015408可能分别以丝裂原激活蛋白激酶激活蛋白激酶2(MA P K-a c t i v a t e d p r o t e i n k i n a s e2,MA P K-A P K2)㊁核受体亚家族1D组成员2(n u c l e a r r e c e p t o r s u b f a m i l y1g r o u p D m e m b e r2, N R1D2)和醛酮还原酶家族1成员C4(a l d o-k e t o r e d u c t a s e f a m i l y1m e m b e rC4,A K R1C4)为靶点,在猪肌内脂肪生成和脂质积累中起重要调节作用; X i n g等[6]研究发现了脂质运载蛋白2(l i p o c a l i n2, L C N2)㊁磷酸烯醇式丙酮酸羧激酶1 (p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e1,P C K1)㊁过氧化物酶体增殖激活受体γ辅激活因子1β(p e r o x i s o m e p r o l i f e r a t i v e a c t i v a t e dr e c e p t o r,g a m m a,c o a c t i v a t o r65828期聂靖茹等:大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析1b e t a,P P A R G C1B)㊁腺苷一磷酸脱氨酶1 (a d e n o s i n em o n o p h o s p h a t ed e a m i n a s e1,AM P D1)和线粒体肌酸激酶2(c r e a t i n ek i n a s e,m i t o c h o n d r i a l 2,C KMT2)5个影响脂肪沉积的候选基因;A n a 等[7]研究发现,R e t i n t o伊比利亚猪脂肪酸结合蛋白3(f a t t y a c i db i n d i n gp r o t e i n3,F A B P3)㊁F A B P5㊁长链脂肪酸转运蛋白1(s o l u t ec a r r i e rf a m i l y27 m e m b e r1,S L C27A1)等基因上调能够加速脂肪沉积,T o r b i s c a l伊比利亚猪脂联素(a d i p o n e c t i n,C1Q a n d c o l l a g e nd o m a i n c o n t a i n i n g,A D I P O Q)和肉毒碱棕榈酰基转移酶1A(c a r n i t i n e p a l m i t o y l t r a n s f e r a s e1A, C P T1A)基因上调会抑制脂肪沉积㊂迪庆藏猪是中国特有的高原型猪种之一,具有沉脂能力强㊁肉质好等特点[8],目前关于迪庆藏猪的研究主要在起源与驯化[9]㊁遗传多样性[10-12]㊁低氧适应[13]㊁肉质[14-16]㊁杂交利用[17]等方面,迪庆藏猪肌内脂肪沉积遗传调控机制尚不明晰㊂本研究采用R N A-S e q技术筛选大型迪庆藏猪不同生长阶段肌内脂肪沉积差异的关键基因并分析其调控途径,以期为大型迪庆藏猪肉品质的遗传改良提供参考㊂1材料与方法1.1试验动物选择胎次相同㊁出生日期及体重相近的纯种大型迪庆藏猪36头,随机分为3组,每组12头,在云南省香格里拉市绿源生态种养专业合作社藏猪养殖基地进行育肥试验,分别在体重达40㊁80㊁120k g左右时屠宰,每组采集3头猪的背最长肌(S1-3L D㊁S2-3L D㊁S3-3L D),液氮速冻运回实验室,-80ħ保存备用㊂1.2主要试剂及仪器动物组织总R N A提取试剂盒㊁反转录-荧光定量试剂盒均购自天根生化科技(北京)有限公司㊂N a n o D r o p2000核酸浓度测定仪购自T h e r m o F i s h e r S c i e n t i f i c公司;荧光定量P C R仪(F Q D-96A)购自杭州博日科技股份有限公司㊂1.3方法1.3.1 R N A提取和质量检测参考王志秀[18]采用T R I z o l法结合动物组织总R N A提取试剂盒提取背最长肌总R N A,去除r R N A㊂利用1.0%琼脂糖凝胶电泳检测所提取总R N A的质量,以确定R N A是否存在降解及污染等情况;利用N a n o D r o p 2000测定仪对总R N A的纯度(D260n m/D280n m)进行测定,利用Q u b i t对总R N A进行精确定量,利用A g i l e n t2100核酸分析仪检测R N A的完整性,检测合格的总R N A用于转录组测序㊂1.3.2去核糖体链特异性文库构建及测序从9个检测合格的样本R N A中各取3μg建库,对测序文库进行质检,文库浓度要求不低于1n g/μL,电泳观察构建的文库条带大小在350~450b p;按照c B o t U s e r G u i d e(P a r t#15006165,R e v.F, I l l u m i n a)流程,在I l l u m i n aH i s e q2000测序仪配套的c B o t上完成C l u s t e r的生成和第一向测序引物杂交;按照H i s e q2000U s e rG u i d e(P a r t#15011190_ H,I l l u m i n a)要求备好测序试剂,将携有c l u s t e r的f l o wc e l l上机,按程序进行P a i r e dE n d2ˑ100n t M u l t i p l e x测序,并进行数据的收集和实时数据分析㊂将m R N A进行O l i g o(d T)磁珠富集,反转录合成c D N A文库㊂使用N l aⅢ内切酶产生标签5'-端,该内切酶可识别并切断c D N A上的C A T G位点,利用磁珠纯化带有c D N A3'-端片段,将其5'-端连接到I l l u m i n a接头1上;I l l u m i n a接头1与C A T G位点的结合处是Mm eⅠ内切酶的识别位点,该内切酶可将识别位点与酶切位点分离,酶切位置在C A T G位点下游17b p处,从而产生带有接头1的T a g;采用聚丙烯酰胺凝胶电泳进行纯化,利用A g i l e n t2100B i o a n a l y z e r和A B IS t e p O n e P l u s R e a l-t i m eP C RS y s t e m对构建好的文库进行质检,质检合格的文库用I l l u m i n aH i s e q2000上机测序㊂1.3.3测序数据处理及差异表达基因聚类分析对测序获得的原始数据(r a wr e a d s),去除污染和低质量片段后获得过滤后的有效数据(c l e a n r e a d s),采用H I S A T2软件将c l e a nr e a d s与猪的参考基因组(S u s s c r o f a11.1)进行比对,对比对到基因组各染色体上的c l e a nr e a d s进行统计,以l o g2| F o l d C h a n g e|>1且P<0.05作为筛选条件,将片段每百万碱基碎片数(f r a g m e n t s p e r k i l o b a s em i l l i o n, F P KM)值归一化进行差异表达基因分析㊂将3个生长阶段分为3个比较组(S1v s S2:40k g v s80k g; S2v s S3:80k g v s120k g;S1v s S3:40k g v s120k g),对每个比较组的阳性共表达基因聚类分析㊂1.3.4差异表达基因功能富集分析对筛选出的差异表达基因进行G O功能和K E G G通路富集分析,筛选与迪庆藏猪脂肪沉积相关的差异表达基因,按富集度c o u n tȡ2且校正P<0.05作为显著富集基因的阈值㊂1.3.5差异表达基因S T E M分析采用短时间序列表达分析(s h o r t t i m e-s e r i e s e x p r e s s i o nm i n e r, S T E M)对差异表达基因进行趋势分析,数据过滤标7582中国畜牧兽医49卷准为3个时间节点两两比较的组内表达差异倍数在2倍以上且P<0.05,对显著差异表达的模块进行G O功能和K E G G通路富集分析㊂1.3.6差异表达基因互作网络分析利用C y t o s c a p e3.9.1软件挖掘差异表达基因间的互作关系,构建差异表达基因的互作网络㊂1.3.7实时荧光定量P C R 用P r i m e rP r e m i e r 5.0软件设计实时荧光定量P C R引物(表1),利用反转录试剂盒将R N A反转录为c D N A,以G A P DH作为内参基因,采用A B I H T7900定量P C R仪对筛选到的差异表达基因进行实时荧光定量P C R检测㊂P C R扩增体系20μL:2ˑT5F a s t q P C R M i x(S Y B R G r e e nⅠ)10μL,上㊁下游引物(10μm o l/L)各0.8μL,c D N A模板1μL,d d H2O 7.4μL㊂P C R扩增条件:95ħ预变性1m i n;95ħ变性15s,退火15s(退火温度见表1),72ħ延伸30s,共40个循环;循环结束后开始熔解曲线分析: 95ħ5s,60ħ1m i n,温度以0.11ħ/s的速率从60ħ递增到95ħ㊂每个样本设3个重复,根据2-ΔΔC t值计算定量结果㊂表1引物信息T a b l e1P r i m e r i n f o r m a t i o n基因G e n e s引物序列P r i m e r s e q u e n c e s(5'ң3')退火温度A n n e a l i n gt e m p e r a t u r e/ħ产物长度P r o d u c tl e n g t h/b pG e n B a n k登录号G e n B a n ka c c e s s i o nN o.A T F3F:T G C T A A C C T G A C A C C C T T T G T60238N C_010451.4R:G C A C T C C G T C T T C T C C T T C T TE G R1F:A G T T T G C C A G G A G C G A T G A A6084N C_010444.4R:A G G C C A C A C T T T T G T C T G C TF O X O1F:A AG A G C G T G C C C T A C T T C A A60180N C_010453.5R:C C T G G G G G A T T T C C C A C T C TL I P E F:C A C A A G T C C C G A C C C A A C T T60239N C_010448.4 R:G G T G C A T C C T C A G G T C G A A AN O R-1F:A A T G C C C T T G T C C G A G C T T T60136N C_010443.5 R:T G G A G G C T G T G A G A A G G T T GP D K4F:G C T G G T G A C T G G T G T A T C C C60141N C_010451.4 R:T C A C A C G C A C A C A T T C A G G AP P A R D F:A G A A C C G C A A C A A G T G C C A G61109N C_010449.5 R:A G C T T C C T T T T C T C T G C C T C GP P A R G C-1F:A A C C C A C A G A G A C C C G A A A C60145N C_010450.4 R:C C C T T G G G G T C A T T T G G T G AP R K A G2F:C A T C G G G A C C T A C G A C A A C A6081N C_010460.4 R:C G C C T T T C C A C G A A G A C G T TS T A T1F:T C C G A C A C C T G C A A C T G A A A60151N C_010457.5 R:G G C A G A G A G G T G G T C T C A A GG A P DH F:T C G G A G T G A A C G G A T T T G60219N C_010447.5R:C C T G G A A G A T G G T G A T G G2结果2.1大型迪庆藏猪背最长肌R N A提取质量及测序数据统计用1.0%琼脂糖凝胶电泳检测总R N A质量,样品总R N A的28S和18S条带清晰,28S的亮度约为18S的2倍,质量达到R N A-S e q上机测序的要求㊂由表2可知,每个样品的r a wr e a d s数都在48M b 以上,c l e a n r e a d s比例均超过88%,有效Q30比例均在94%以上,比对率均在95%以上,测序数据符合生物信息学分析要求㊂85828期聂靖茹等:大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析表2 R N A -S e q 测序数据统计T a b l e 2 R N A -S e q d a t a s t a t i s t i c s 样品S a m p l e s 原始数据R a wr e a d s /b p 有效数据C l e a n r e a d s /b p 有效数据比例C l e a n r e a d s r a t e/%有效Q 30比例C l e a nQ 30b a s e s r a t e/%比对率M a p p i n g ra t i o /%S 1-1L D 499858864691529693.8694.5996.17S 1-2L D 508196444590462890.3394.3795.94S 1-3L D 484290644422050091.3194.5195.76S 2-1L D 493129684616038693.6194.6996.17S 2-2L D 538149884779656088.8294.8896.17S 2-3L D 512092004798930493.7194.7996.36S 3-1L D 515240764792055693.0194.5596.06S 3-2L D 512190064687473691.5294.5095.50S 3-3L D 493982364597920693.0894.4696.062.2 大型迪庆藏猪背最长肌差异表达基因聚类分析对3个比较组的阳性共表达基因进行聚类发现,每个组内部聚为一类,组间明显分开(图1),可进行后续分析㊂A ,S 1v s S 2;B ,S 2v s S 3;C ,S 1v s S 3㊂图2同A ,S 1v s S 2;B ,S 2v s S 3;C ,S 1v s S 3.T h e s a m e a s f i g .2图1 大型迪庆藏猪背最长肌差异表达基因聚类热图F i g .1 C l u s t e r i n g h e a tm a p o f d i f f e r e n t i a l l y e x p r e s s e d g e n e s i n l o n g i s s i m u s d o r s im u s c l e o f l a r g eD i q i n g T i b e t a n p i gs 2.3 不同体重阶段大型迪庆藏猪背最长肌显著差异基因筛选以l o g 2|F o l d C h a n g e |>1且P <0.05为筛选显著差异基因的阈值,S 1v s S 2阶段共筛选到730个基因显著差异表达,其中,上调基因341个,下调基因389个(图2A );S 2v s S 3阶段共筛选到981个基因显著差异表达,其中,530个基因上调,451个基因下调(图2B );S 1v s S 3阶段共筛选到735个基因显著差异表达,其中,364个基因上调,371个基因下调(图2C)㊂2.4 不同体重阶段大型迪庆藏猪背最长肌显著差异表达基因功能富集分析S 1v s S 2阶段,G O 功能分析结果显示,差异表达基因主要富集在凋亡过程调控㊁胰岛素应答㊁脂肪细胞分化等过程(图3A );K E G G 通路分析结果显示,差异表达基因主要富集在F O X O 信号通路㊁脂肪细胞因子信号通路㊁胰岛素抵抗等通路(图3B )㊂S 2v s S 3阶段,G O 功能分析结果显示,差异表达基因主要富集在葡萄糖稳态㊁脂肪垫发育㊁脂肪酸氧化正调控等过程(图3C );K E G G 通路分析结果显示,差异表达基因主要富集在胰岛素信号通路㊁P P A R 信号通路㊁脂肪细胞因子信号通路等(图3D )㊂S 1v s S 3阶段,G O 功能富集分析显示,差异表达基因主要富集在葡萄糖应答㊁脂肪细胞分化㊁能量稳态等过程(图3E );K E G G 通路富集分析显示,差异表达基因主要富集在胰岛素信号通路㊁胰岛素抵抗信号通路等(图3F )㊂9582中国畜牧兽医49卷图2 大型迪庆藏猪背最长肌差异表达基因火山图F i g .2 V o l c a n i cm a p o f d i f f e r e n t i a l l y e x p r e s s e d g e n e s i n l o n g i s s i m u s d o r s im u s c l e o f l a r g eD i q i n g T i b e t a n p i gs 06828期聂靖茹等:大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析A ㊁C ㊁E ,S 1v s S 2㊁S 2v s S 3和S 1v s S 3G O 功能富集图;B ㊁D ㊁F ,S 1v s S 2㊁S 2v s S 3和S 1v s S 3K E G G 通路富集图A ,Ca n dE ,G Of u n c t i o ne n r i c h m e n t h i s t o g r a m so fS 1v s S 2,S 2v s S 3a n dS 1v s S 3,r e s p e c t i v e l y ;B ,Da n dF ,K E G G p a t h w a y e n r i c h m e n t h i s t o g r a m s o f S 1v s S 2,S 2v s S 3a n dS 1v s S 3,r e s p e c t i v e l y图3 大型迪庆藏猪背最长肌差异表达基因G O 功能和K E G G 通路富集分析图F i g .3G Of u n c t i o na n dK E G G p a t h w a y e n r i c h m e n th i s t o g r a m so fd i f f e r e n t i a l l y e x p r e s s e d g e n e s i nl o n g i s s i m u sd o r s im u s c l eo f l a r g eD i q i n g T i b e t a n p i gs 2.5 大型迪庆藏猪背最长肌差异表达基因S T E M 分析对筛选到的显著差异表达基因进行S T E M 分析,共有4个差异显著模块(图4),模块11和14的差异表达基因聚为一类,先表达上调后呈现轻微表达下调;模块9和10的差异表达基因聚为一类,先表达上调后表达下调;其余模块差异不显著㊂模块11和14的基因集G O 功能分析显示,差异表达基因主要富集到B M P 信号通路的负调控㊁J U N 激酶活性激活等过程(图5A ),K E G G 通路分析显示,差异表达基因主要富集到P I 3K -A k t 信号通路㊁E C M 受体相互作用等通路(图5B );模块9和10基因集G O 功能主要富集到细胞对脂多糖反应㊁T 细胞凋亡过程的正调控(图5C ),K E G G 通路主要富集到Ⅱ型糖尿病㊁T 细胞受体信号通路等通路(图5D )㊂颜色背景表示趋势显著(P <0.05),相同颜色表示模块内所包含的基因表达趋势相似;白色背景模块表示趋势不显著(P >0.05)T h e t r e n d o f c o l o r b a c k g r o u n d i s s i g n i f i c a n t (P <0.05),t h e s a m e c o l o r i n d i c a t e s t h a t t h e g e n e e x p r e s s i o n t r e n d c o n t a i n e d i n t h e m o d u l e i s s i m i l a r ;T h e t r e n do fw h i t eb a c k g r o u n dm o d u l e i sn o t s i g n i f i c a n t (P >0.05)图4 大型迪庆藏猪背最长肌S T E M 分析F i g .4 S T E Ma n a l y s i s o f l o n g i s s i m u s d o r s im u s c l e i n l a r g eD i q i n g T i b e t a n p i gs 1682中国畜牧兽医49卷A ㊁B ,模块11㊁14的G O 功能和K E G G 通路富集图;C ㊁D ,模块9及10的G O 功能和KE G G 通路富集图Aa n dB ,G Of u n c t i o na n dK E G G p a t h w a y e n r i c h m e n t h i s t o g r a m s o fm o d u l e 11a n d14,r e s p e c t i v e l y ;Ca n dD ,G Of u n c t i o na n d K E G G p a t h w a y e n r i c h m e n t h i s t o g r a m s o fm o d u l e 9a n d10,r e s p e c t i v e l y图5 大型迪庆藏猪背最长肌差异表达基因显著富集模块G O 功能和K E G G 通路富集分析图F i g .5G Of u n c t i o na n dK E G G p a t h w a y e n r i c h m e n t h i s t o g r a m s o f d i f f e r e n t i a l l y e x p r e s s e d g e n e s o f s i g n i f i c a n t e n r i c h m e n tm o d u l e s i n l o n g i s s i m u s d o r s im u s c l e o f l a r g eD i q i n g T i b e t a n p i gs 2.6 不同体重阶段大型迪庆藏猪背最长肌差异表达基因互作网络2.6.1 S 1v s S 2 选择S 1v s S 2阶段显著富集通路中的基因绘制网络图,结果见图6㊂由图6可知,转录激活因子3(a c t i v a t i n g t r a n s c r i pt i o nf a c t o r3,A T F 3)㊁核受体亚家族4A 组成员3(n u c l e a rr e c e p t o r s u b f a m i l y 4g r o u p A m e m b e r3,N R 4A 3/N O R -1)㊁AM P 激活蛋白激酶γ2调节亚单位(p r o t e i n k i n a s e A M P -a c t i v a t e d n o n -c a t a l yt i cs u b u n i t g a m m a 2,P R K A G 2)㊁早期生长应答因子1(e a r l y g r o w t hr e s po n s e1,E G R 1)㊁早期生长应答因子2(e a r l yg r o w t h r e s p o n s e2,E G R 2)基因位于网络核心,其中A T F 3㊁N O R -1㊁P R K A G 2基因表达上调,A T F 3基因与脂联素表达有关,N O R -1和P R K A G 2基因会影响胰岛素敏感性;E G R 1㊁E G R 2基因表达下调,这2个基因与脂质代谢㊁脂肪生成有关㊂26828期聂靖茹等:大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析图6 S 1v s S 2阶段差异表达基因互作网络F i g .6 I n t e r a c t i o n n e t w o r k d i a g r a mo f d i f f e r e n t i a l l y e x p r e s s e d g e n e s a t S 1v s S 2s t a ge 2.6.2 S 2v s S 3 选择S 2v s S 3阶段显著富集通路中的基因绘制网络图,结果见图7㊂由图7可知,过氧化物酶体增殖物激活受体δ(pe r o x i s o m e p r o l if e r a t o r a c t i v a t e d r e c e p t o r d e l t a ,P P A R D )㊁丙酮酸脱氢酶激酶(p y r u v a t ed e h y d r og e n a s ek i n a s e4,P D K 4)㊁叉头盒O 1(f o r kh e a db o xO 1,F O X O 1)㊁过氧化物酶体增殖激活受体γ辅激活因子1α(pe r o x i s o m e p r o l if e r a t i v ea c t i v a t e dr e c e p t o r ,ga m m a ,c o a c t i v a t o r 1a l p h a ,P P A R G C 1A /P P A R G C -1)㊁A T F 3㊁信号转导和转录激活因子1(s i gn a l t r a n s d u c e r a n da c t i v a t o r o f t r a n s c r i p t i o n 1,S T A T1)㊁脂肪酶E (l i pa s eE ,L I P E )基因位于网络中心,其中,L I P E ㊁A T F 3基因表达上调,L I P E 基因参与抗脂分解㊁脂质稳态等过程,A T F 3基因可调控脂联素表达;P P A R D ㊁P D K 4㊁F O X O 1㊁P P A R G C -1㊁S T A T 1基因表达下调,P P A R D 基因在脂肪代谢中发挥作用,P D K 4和F O X O 1基因可相互作用影响葡萄糖摄取及游离脂肪酸含量,P P A R G C -1基因在脂肪储存中起调控作用,S T A T 1基因可在脂肪细胞分化过程中起作用㊂2.6.3 S 1v s S 3 选择S 1v s S 3阶段显著富集通路中的基因绘制基因网络图,结果见图8㊂由图8可知,A T F 3㊁N O R -1㊁E G R 1㊁E G R 2㊁S T A T 1基因位于网络中心,其中,A T F 3㊁N O R -1基因表达上调,A T F 3基因可调控脂联素表达,N O R -1基因影响胰岛素敏感性;E G R 1㊁E G R 2㊁S T A T 1基因表达下调,E G R 1㊁E G R 2基因影响脂质代谢,S T A T 1基因与脂肪细胞分化有关㊂图7 S 2v s S 3阶段差异表达基因互作网络F i g .7 I n t e r a c t i o n n e t w o r k d i a g r a mo f d i f f e r e n t i a l l y e x p r e s s e d g e n e s a t S 2v s S 3s t a ge 图8 S 1v s S 3阶段差异表达基因互作网络F i g .8 I n t e r a c t i o n n e t w o r k d i a g r a mo f d i f f e r e n t i a l l y e x p r e s s e d g e n e s a t S 1v s S 3s t a ge 2.7 大型迪庆藏猪背最长肌脂肪沉积差异表达基因实时荧光定量验证随机挑选了E G R 1㊁F O X O 1等10个差异表达基因进行实时荧光定量P C R 验证,E G R 1㊁F O X O 1㊁P D K 4㊁P P A R D ㊁P P A R G C -1㊁S T A T 1基因表达下调,A T F 3㊁L I P E ㊁N O R -1㊁P R K A G 2基因表达上调,与R N A -S e q 结果一致(图9)㊂3682中国畜牧兽医49卷图9 大型迪庆藏猪背最长肌差异表达基因实时荧光定量P C R 验证结果F i g .9 R e a l -t i m e q u a n t i t a t i v eP C Rv e r i f i c a t i o n r e s u l t s o f d i f f e r e n t i a l l y e x p r e s s e d g e n e s i n l o n g i s s i m u s d o r s im u s c l e o f l a r g eD i q i n g T i b e t a n p i gs 3 讨论3.1 大型迪庆藏猪S 1v s S 2背最长肌脂肪沉积差异表达基因大型迪庆藏猪40k g v s 80k g 阶段,A T F 3㊁N O R -1㊁P R K A G 2基因表达上调,E G R 1㊁E G R 2基因表达下调㊂A T F 3㊁N O R -1和P R K A G 2基因均在胰岛素抵抗信号通路中富集,当诱发胰岛素抵抗时,脂肪细胞㊁肌肉细胞对正常浓度的胰岛素反应不足,使得胰岛素作用的敏感性降低,促进葡萄糖摄取和利用效率下降,能量摄入过多[19],脂肪分解减少[20]㊂而脂联素能够激活腺苷酸活化蛋白激酶(AM P -a c t i v a t e d p r o t e i nk i n a s e ,AM P K ),减少丙二酰辅酶A 生成,促使脂肪酸进入线粒体得到氧化[21]㊂当发生脂联素抵抗时,脂联素对AM P K 的刺激减弱,骨骼肌中脂肪酸氧化的作用减弱,从而促进胰岛素抵抗[22]㊂A T F 3基因是脂联素基因表达的负调节因子,脂联素基因低表达会导致肥胖和胰岛素抵抗[23]㊂N O R -1基因在骨骼肌和脂肪组织中大量表达,参与肌内脂肪形成[24],N O R -1基因过度表达会导致循环儿茶酚胺浓度降低,引起胰岛素敏感性降低[25],导致脂肪分解减少,脂肪沉积增加[20]㊂P R K A G 2基因编码AM P 活化蛋白激酶γ2亚单位,该基因上调导致AM P K 活性增加,糖原合成激活㊁葡萄糖摄取增加,诱发胰岛素抵抗,脂肪沉积增多[26]㊂本试验中,A T F 3基因表达上调,引起脂联素基因下调,可能导致脂肪含量增加;N O R -1基因上调引起胰岛素敏感性降低,导致脂肪分解减少;P R K A G 2基因上调导致糖原合成激活㊁葡萄糖摄取增加,脂肪沉积增多㊂E G R 1和E G R 2基因属于早期生长反应因子家族,与脂质代谢㊁脂肪生成㊁胰岛素及胆固醇生物合成有关,在脂肪细胞分化信号通路中富集㊂E G R 1基因是3T 3-L 1脂肪细胞分化的负调节因子,敲除E G R 1基因能增强脂肪细胞分化[27]㊂E G R 2基因是脂肪细胞分化负调节因子m i R -224-5p 的直接靶点,m i R -224-5p 在早期脂肪形成中调节EG R 2基因表达[28]㊂本试验中,E G R 1和E G R 2基因表达下调,可能促进脂肪细胞分化和脂肪形成㊂3.2 大型迪庆藏猪S 2v s S 3背最长肌脂肪沉积差异表达基因大型迪庆藏猪80k g v s 120k g 阶段,L I P E ㊁A T F 3基因表达上调,P P A R D ㊁P D K 4㊁F O X O 1㊁P P A R G C -1㊁S T A T 1基因表达下调㊂L I P E 和A T F 3基因均富集到葡萄糖稳态信号通路中,影响肌肉组织对葡萄糖的吸收与利用及脂肪组织对胰岛素的敏感性[29]㊂L I P E 基因参与游离脂肪酸的动员[30]㊁抗脂分解及脂质稳态,导致高肌内脂肪含量[31]㊂A T F 3基因通过负调控脂联素基因而引起肥胖和胰岛素抵抗[23]㊂本试验中,L I P E 基因表达上调可能减少脂肪分解㊁提高肌内脂肪含量,A T F 3基因表达上调可引起脂联素基因表达下调,可能导致肌内脂肪含量增加㊂P P A R D 基因在W n t 和P P A R 信号通路中富集,参与脂肪代谢㊁能量调控,进而影响脂肪细胞的分化[32]㊂研究发现,W n t 信号通路能够调节脂肪沉积[33],同时能维持前脂肪细胞向脂肪成熟细胞分化的状态,W n t 信号通路减弱时,可分化大量脂肪细胞[34]㊂P P A R D 基因是胰岛素敏感和脂肪代谢的关键调节因子,在脂肪代谢中发挥作用,主要参与脂肪酸分解[35],增强脂肪酸运输㊁氧化㊁能量解偶联㊁线粒体呼吸㊁产热等相关基因的转录[36],上调脂肪酸氧化相关基因,降低体重㊁脂滴数量和脂滴大46828期聂靖茹等:大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析小[35]㊂猪P P A R D基因定位在7号染色体一个脂肪沉积的数量性状基因座(q u a n t i t a t i v e t r a i t l o c u s, Q T L)附近,可促进胆固醇和血清高密度脂蛋白积累[37]㊂P D K4㊁F O X O1㊁P P A R G C-1㊁S T A T1基因均在葡萄糖稳态㊁胰岛素受体及胰岛素信号通路过程中富集,通过影响脂联素及胰岛素作用的敏感性,进而影响葡萄糖摄取和利用效率及能量代谢,从而影响肌内脂肪含量㊂F O X O1基因会对脂联素受体的表达及脂联素敏感性的调节产生影响[38],同时F O X O1基因在调节胰岛素信号转导的糖异生和糖原分解中发挥重要作用,通过抑制细胞周期蛋白依赖激酶而阻止脂肪生成转录因子表达[39],在终末分化开始时,F O X O1基因被激活并转移到前脂肪细胞的细胞核,与P P A R G基因的启动子结合,导致有丝分裂后细胞生长停滞,负调控脂肪生成[40]㊂此外,F O X O1与P D K4基因的启动子区结合,增加肌肉中P D K4基因m R N A的表达[41],减少丙酮酸脱氢酶复合物(p y r u v a t e d e h y d r o g e n a s e c o m p l e x, P D C)通量,减弱碳水化合物氧化和葡萄糖摄取,并减少游离脂肪酸[42]㊂P P A R G C-1是一种转录共激活因子,可调控能量代谢,在米色和棕色脂肪组织中对脂肪代谢㊁脂肪细胞增殖㊁脂肪储存起重要调控作用[43],脂肪组织中缺乏P P A R G C-1基因的小鼠会产生胰岛素抵抗[44]㊂S T A T1基因位于P P A R G基因下游,在脂肪细胞分化中通过级联调节负调控脂肪形成[45]㊂本试验中,P P A R D㊁F O X O1㊁P P A R G C-1㊁S T A T1㊁P D K4基因表达下调, P P A R D基因表达下调可能使脂肪酸运输㊁氧化等相关基因的转录下调,脂肪酸分解减少㊁脂肪酸积累增多㊁脂滴变大㊂F O X O1基因表达下调导致与P P A R G基因的启动子结合减少,脂肪生成增多;另外,F O X O1与P D K4基因的启动子结合减少,葡萄糖摄取增加,游离脂肪酸增多㊂P P A R G C-1基因表达下调可能使骨骼肌对胰岛素的敏感性降低,肌内脂肪含量增加㊂S T A T1基因位于P P A R G基因下游,P P A R G基因通过级联调控引起S T A T1基因表达下调,促进脂肪生成㊂3.3大型迪庆藏猪S1v s S3背最长肌脂肪沉积差异表达基因大型迪庆藏猪40k g v s120k g阶段,A T F3㊁N O R-1基因表达上调,S T A T1㊁E G R1㊁E G R2基因表达下调㊂A T F3和N O R-1基因富集到胰岛素信号通路中,通过影响肌肉组织和脂肪组织中胰岛素敏感性来影响葡萄糖摄取及能量代谢效率,增加肌内脂肪含量[19-20]㊂S T A T1基因在能量稳态㊁胰岛素抵抗信号通路中富集,通过级联调节负调控脂肪形成[45]㊂E G R1㊁E G R2基因属于立早基因(i m m e d i a t e e a r l yg e n e s,I E G s),参与细胞生长㊁分化,富集到脂肪细胞分化信号通路㊂E G R1和E G R2为脂肪细胞分化的负调节因子[27-28],其表达下调促进脂肪形成㊂4结论A T F3㊁N O R-1㊁P R K A G2㊁E G R1㊁E G R2㊁L I P E㊁P P A R D㊁P D K4㊁F O X O1㊁P P A R G C-1㊁S T A T1作为核心基因调控大型迪庆藏猪肌内脂肪沉积;不同生长阶段参与调控的核心基因并不完全相同,40k g v s80k g阶段,A T F3㊁N O R-1㊁P R K A G2基因表达上调,E G R1㊁E G R2基因表达下调;80k g v s 120k g阶段,L I P E㊁A T F3表达上调,P P A R D㊁P D K4㊁F O X O1㊁P P A R G C-1㊁S T A T1基因表达下调;40k g v s120k g阶段,A T F3㊁N O R-1基因表达上调,E G R1㊁E G R2㊁S T A T1基因表达下调㊂参考文献(R e f e r e n c e s):[1] WO O DJ D,E N S E R M,F I S H E R A V,e ta l.F a td e p o s i t i o n,f a t t y a c i dc o m p o s i t i o na n dm e a t q u a l i t y:Ar e v i e w[J].M e a t S c i e n c e,2008,78:343-358. 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rf a tr e l a t e dt ol i p i d m e t a b o l i s m i nt w o p ig b r e e d s[J].C e l l u l a rP h y s i o l o g y a n dB i o c h e m i s t r y,2018,50(6):2406-2422.5682中国畜牧兽医49卷[6] X I N G K,WA N G K J,A O H,e ta l.C o m p a r a t i v ea d i p o s e t r a n s c r i p t o m ea n a l y s i sd i g so u t g e n e sr e l a t e dt of a t d e p o s i t i o ni n t w o p i g b r e e d s[J].S c i e n t i f i cR e p o r t s,2019,9(1):12925.[7] A N A V,L U I S N J,N A T A C HA P R,e t a l.C o m p a r a t i v e t r a n s c r i p t o m e p r o f i l e b e t w e e n I b e r i a n p i gv a r i e t i e s p r o v i d e sn e wi n s i g h t s i n t ot h e i rd i s t i n c t f a td e p o s i t i o na n d f a t t y a c i d s c o n t e n t[J].A n i m a l s,2021,11(3):627.[8]云南省畜牧局,云南省家畜家禽品种志编写委员会.云南省家畜家禽品种志[M].昆明:云南科技出版社,1987.A N I MA L HU SB A N D R Y B U R E A U O F Y U N N A NP R O V I N C E,C OM P I L A T I O N C OMM I T T E E O FL I V E S T O C K A N D P O U L T R Y B R E E D S O FY U N N A N P R O V I N C E.S p e c i e s o f L i v e s t o c k a n dP o u l t r y i n Y u n n a n P r o v i n c e[M].K u n m i n g:Y u n n a nS c i e n c e a n dT e c h n o l o g y P r e s s,1987.(i nC h i n e s e) [9] Y A N GS L,Z H A N G H,M A O H M,e ta l.T h el o c a lo r i g i n o f t h eT i b e t a n p i g a n da d d i t i o n a l i n s i g h t s i n t o t h eo r i g i n o fA s i a n p i g s[J].P L o SO n e,2011,6(12):e28215.[10]李相运,任战军,常洪.迪庆藏猪的遗传多样性研究[J].生物多样性,2000,8(3):253-256.L IX Y,R E N ZJ,C HA N G H.G e n e t i cd i v e r s i t y o fD i q i n g-T i b e t a n p i g o fY u n n a n[J].C h i n e s eB i o d i v e r s i t y,2000,8(3):253-256.(i nC h i n e s e)[11]李相运,常洪.合作猪㊁迪庆藏猪和成华猪的血清运铁蛋白多型性[J].甘肃农业大学学报,2000,35(2):194-196.L IX Y,C HA N G H.T r a n s f e r r i n p o l y m o r p h i s m o fH e z u o p i g a n dD i q i n g-T i b e t a n p i g a n dC h e n g h u a p i g i nC h i n a[J].J o u r n a lo f G a n s u A g r i c u l t u r a l U n i v e r s i t y,2000,35(2):194-196.(i nC h i n e s e)[12]方晨,胡瑞举,杨明华,等.迪庆藏猪F U T1基因遗传变异分析[J].中国畜牧杂志,2020,56(12):29-35.F A NG C,HU R J,Y A N G M H,e t a l.G e n e t i cv a r i a t i o na n a l y s i so f F U T1g e n ei n D i q i n g T i b e t a np i g[J].C h i n e s eJ o u r n a lo f A n i m a l S c i e n c e,2020,56(12):29-35.(i nC h i n e s e)[13] WU DD,Y A N GCP,WA N G M S,e t a l.C o n v e r g e n tg e n o m i c s i g n a t u r e so fh i g h-a l t i t u d ea d a p t a t i o na m o n gd o me s t i c m a m m a l s[J].N a t i o n a l S c i e n c e R e v i e w,2020,7(6):952-963.[14]严达伟,赵桂英,苟潇,等.迪庆藏猪肉质特性的研究[J].云南农业大学学报,2007,22(1):86-91.Y A N D W,Z H A O G Y,G O U X,e ta l.S t u d y o nc h a r a c t e r i s t i c s o fD i q i n g T i b e t a n p i g sm e a t q u a l i t y[J].J o u r n a lo f Y u n n a n A g r i c u l t u r a l U n i v e r s i t y,2007,22(1):86-91.(i nC h i n e s e)[15]马黎,聂靖茹,鲁绍雄,等.迪庆藏猪与野藏杂交猪肌肉全谱游离氨基酸味道强度值比较[J].中国畜牧兽医,2021,48(4):1275-1283.MA L,N I EJR,L U S X,e ta l.C o m p a r i s o no ft h et a s t e a c t i v i t y v a l u e o f f u l l s p e c t r u mf r e e a m i n o a c i d i nm u s c l e o fD i q i n g T i b e t a n p i g a n d W i l dB o a rˑD i q i n gT i b e t a n p i g[J].C h i n aA n i m a lH u s b a n d r y&V e t e r i n a r yM e d i c i n e,2021,48(4):1275-1283.(i nC h i n e s e) [16]马黎,张博,鲁绍雄,等.大型迪庆藏猪与野藏杂交猪肌肉全谱游离氨基酸比较[J].江西农业大学学报,2020,42(5):941-952.MAL,Z HA N GB,L U SX,e t a l.C o m p a r i s o no f f u l ls p e c t r u mf r e ea m i n oa c i di n m u s c l eo fl a r g e D i q i n gT i b e t a n p i g a n dh y b r i d W i l db o a rˑT i b e t a n p i g[J].A c t aA g r i c u l t u r a lU n i v e r s i t a t i sJ i a n g x i e n s i s,2020,42(5):941-952.(i nC h i n e s e)[17]刘韶娜,张斌,相德才,等.迪庆藏猪杂交后代生长性能及屠宰性能比较研究[J].江苏农业科学,2020,48(19):181-184.L I USN,Z HA N GB,X I A N GDC,e t a l.C o m p a r a t i v es t u d y o n g r o w t h p e r f o r m a n c e a n ds l a u g h t e r p e r f o r m a n c eo f D i q i n g T i b e t a n p i g h y b r i d o f f s p r i n g[J].J i a n g s uA g r i c u l t u r a l S c i e n c e s,2020,48(19):181-184.(i nC h i n e s e)[18]王志秀.基于转录组和蛋白组数据鉴定猪肌肉生长和脂肪沉积相关的基因[D].北京:中国农业大学,2017.WA N GZX.I d e n t i f i c a t i o no f g e n e s r e l a t e dt om u s c l eg r o w t ha n dl i p i dd e p o s i t i o nf r o mt r a n s c r i p t o m i ca n dp r o t e o m i c p r o f i l e s i n p i g[D].B e i j i n g:C h i n aA g r i c u l t u r a lU n i v e r s i t y,2017.(i nC h i n e s e)[19] L E B O V I T Z H E.I n s u l i nr e s i s t a n c e:D e f i n i t i o n a n dc o n s e q u e n c e s[J].E x p e r i m e n t a l a nd C l i n i c a lE n d o c r i n o l o g y&D i a b e t e s,2001,109(S u p p l2):S135-S148.[20] K I M J Y,N A S R A,T F A Y L I H,e ta l.I n c r e a s e dl i p o l y s i s,d i m i n i s h e d a d i p o s e t i s s u e i n s u l i n s e n s i t i v i t y,a n di m p a i r e db e t a-c e l lf u n c t i o n r e l a t i v et o ad i p o s et i s s u e i n s u l i n s e n s i t i v i t y i no b e s e y o u t hw i t h i m p a i r e dg l u c o s et o l e r a n c e[J].D i a b e t e s,2017,66(12):3085-3090.6682。

Agilent 2100 Bioanalyzer 说明书

Bioanalizzatore Agilent 2100Our measure is your success.possibilità per l’analisi di DNA,RNA, proteineproducts | applications | software | servicesBioanalizzatore Agilent 2100 Tecnologia lab-on-a-chip applicataIl bioanalizzatore Agilent 2100 rappresenta lo standard di riferimento per il QC di campioni di RNA e ha sostituito la piùimpegnativa elettroforesi su gel per questa applicazione. Inoltre, sta rapidamente sostituendo la stessa tecnica anche per l’analisi di frammenti di DNA e della tecnica SDS-PAGE per l'analisi delle proteine. Una caratteristica esclusiva del bioanalizzatore Agilent 2100 è la possibilità di utilizzarlo sia per le separazioni elettroforetiche sia per le analisi citometriche a flusso dei parametri di fluorescenza cellulare. Questa versatilità rende il Bioanalizzatore Agilent 2100 uno strumento indispensabile per la biologia e la biochimica miniaturizzazione della strumentazione analitica presenta numerosi vantaggi rispetto alle tecniche tradizionali. Questi vantaggi comprendono il miglioramento della precisione e della riproducibilità dei dati, i tempi di analisi più corti, il consumo minimo di campione, il potenziamento dell’automazione e l'integrazione di flussi operativi complessi. Introdotto nel 1999, il Bioanalizzatore Agilent 2100è stato il primo strumento commerciale a usare la tecnologia microfluidica per l’analisi di campioni biologici.Possibilità di scelta fra due configurazioni:•Bioanalizzatore Agilent 2100 con cartucce intercambiabili, per applicazioni in elettroforesi e in citometria a flusso•Bioanalizzatore per elettroforesi Agilent 2100 con cartucce intercambiabili, solo per applicazioni in elettroforesi Per entrambi i sistemi la configurazione include:1. Stazione di riempimento chip2. Vortex per chip3. Software Agilent 2100 expert per il controllo dellostrumento e l’elaborazione dei dati (espandibile per un completo supporto alla conformità negli ambientiregolamentati)4. Assistenza e supporto su sistemi e applicazioni23•Citometria a flusso su chipIl modo più semplice per acquisire dati di fluorescenza cellulare a doppia colorazione•Controllo della qualità dell’RNA con RINLo standard dell’industria per l’analisi dell’RNA che fornisce dati di RNA totale, mRNA e Small RNAcompreso l’algoritmo per il RIN (RNA Integrity Number)Un’ampia gamma di applicazioniUn unico sistema compatto con kit pronti per l’uso•Dimensione e quantità del DNASeparazione e quantificazione del DNA di alta qualità•Sostituzione SDS-PAGE per l’analisi delle proteine.Il modo veloce ed affidabile per determinare la quantità e la purezza delle proteine in base alla sensibilità della macchiatura d’argento CoomassieIl bioanalizzatore Agilent 2100 offre un’ampia gamma di kit di analisi pre-validati combinati con un sistema da banco di facile utilizzo Bioanalizzatore Agilent 2100: analisi rapide ed eccellente qualità dei dati•Test pronti per l’uso e kit di reagenti preconfezionati •Minimo consumo di campione (1-4 µl) e risultati entro 30minuti•Cartuccia elettrodo sostituibile per cambiare metodo senza rischio di contaminazioni•Migliore accuratezza e precisione dell’analisi •Risultati interlaboratorio confrontabili•Dati digitali per semplificare le operazioni di analisi,archiviazione e memorizzazione•Opzioni per la visualizzazione dei dati come immagine gel,elettroferogrammi e tabelle•Facilità d'uso con confronto dei campioni semplificato •Minima esposizione a materiali pericolosi •Supporti per la conformità al CFR 21 Parte 11Small RNAPurezza proteine4Facile da utilizzareConfronto dei campioni semplificato•Kit di reagenti pronti all’uso •Istruzioni per un rapido avvio dell'analisi•Preparazione del chip in meno di cinque minuti•Minimo impiego di sostanzechimiche tossiche e quantità minime di rifiuti da smaltire•Volumi di campione nel range dei µlIl campione si sposta attraverso imicrocanali partendo dal pozzetto del campione.Il campione viene iniettato nel canaledi separazione.I componenti del campione vengonoseparati elettroforeticamente.I componenti vengono rivelatimediante la loro fluorescenza e convertiti in immagini gel simili(bande) ed elettroferogrammi (picchi).Principio di funzionamento del lab-on-a-chip1.3.4.2.Ottieni il massimo dai tuoi dati digitaliUn software potente per l’analisi di RNA, DNA, proteine e celluleIl software Agilent 2100 Expert è l’unica soluzione creata specificamente per il sistema bioanalizzatore. Costruito sull’esperienza di numerose generazioni di software per bioanalizzatore, offre la flessibilità della piattaforma integrata 2100 expert, con funzioni accuratamente progettate per aiutarti a ottenere il massimo dai dati digitali. Funzioni dedicate•Strumenti potenti per la valutazione dei dati: un’unica piattaforma per tutte le analisi•Algoritmo esclusivo per il numero di integrità dell’RNA (RIN) per una stima dell’integrità dell’RNA totale senza errori.•Pronto per la conformità - servizi di supporto per la valutazione del sistema (IQ e OQ) e conformità al CFR 21 Parte 11 usando il Security pack opzionale.•Segnalazione sui risultati con codice colore – un sistema di facile uso, con regole definite per il QC e la valutazione dei dati•T abelle e grafici flessibili dei risultati permettono un facile controllo dello strumento e supportano modalità utente standard e avanzate•Funzioni di sovrapposizione, regolazione e zoom con un solo clic del mouse consentono di confrontare velocemente fino a 48 campioni presenti in uno o in vari chip •Molteplici formati di esportazione per un flessibile scambio di dati•Il software di revisione dei dati permette la valutazione e la condivisione offline•Efficienti strumenti integrati per la diagnostica minimizzano i tempi di fermo del sistema•Migliori analisi di campioni di RNA, DNA e proteine •File in formato XML nativo per l’integrazione totale dei dati fra piattaforme e progettiImposta e avviaAnalizza e confrontaLe varievisualizzazionigrafiche, comel’immagine gelsimile quimostrata, sonosupportate databelle diinformazioniaggiuntive eagevolano l’analisidei risultati.Impostazione deicampioni, sceltadel metodo e avviodell’analisi sonocomodamentedisponibili tramiteun’interfacciautente intuitiva chefornisce l’accessoagli strumenti dellaserie 2100.Stampa i reportI report possonoessere configuratifacilmente dagliutenti per soddisfarele loro esigenzesegnalazione deirisultati tramitecodice coloreconsente agli utentidi vedereimmediatamente sei risultati sono entroi limiti.56Agilent supporta il tuo processo di validazione dall’inizio alla finePer gli utilizzatori che lavorano in ambienti regolamentati, il software Agilent 2100Expert supporta le procedure di IQ e OQ/PV di hardware e software, in un contesto di validazione dedicato.Qualificazione del design (DQ)La procedura di DQ definisce le specifiche funzionali e operative del Bioanalizzatore 2100 assicurando la presenza delle funzioni necessarie e dei criteri di prestazione. I documenti forniti per il Bioanalizzatore 2100 comprendono:•Dichiarazione di validazione del sistema•Dichiarazione di conformità dello strumento alle specifiche di fabbricazioneGli strumenti e i software Agilent spianano la via alla conformitàLe aziende biofarmaceutiche devono sopportare i costi e il carico di lavoro supplementari associati al controllo della qualità e alla conformità alle normative. Per questo, le procedure di laboratorio e di produzione devono essere standardizzate e al tempo stesso soddisfare le richieste di GLP e GMP. Agilent ti aiuta a garantire la sicurezza del cliente, minimizzando l’impatto per l’azienda, con il Bioanalizzatore Agilent 2100, per un controllo qualitàstandardizzato e affidabile. Agilent supporta il processo di validazione fornendo la documentazione di DQ e IQ e offrendo strumenti e servizi di OQ/PV. Il software Security Pack del Bioanalizzatore Agilent 2100 risponde a tutti i requisiti del CFR 21 Parte 11 relativi al trattamento delle registrazioni elettroniche, alla sicurezza e all’integrità dei dati e alla tracciabilità per l’audit. Il Security Pack richiede il software 2100 Expert.•Dichiarazione di conformità dello strumento ai requisiti della Guida 22ISO/IEC e EN 45014 CEN/CENELEC •Dichiarazione di conformità per chip e reagenti.Qualificazione dell’installazione (IQ)La procedura di IQ assicura che il Bioanalizzatore Agilent 2100 e il software 2100 expert sono statiinstallati in modo corretto al momento della consegna.“Sono convinto che Agilent fornisca la gamma piùcompleta di servizi per laconformità e la validazione, con pacchetti globali, multi-vendor e multiprodotto, che ticonsentono di risolvere con una chiamata ogni tua esigenza.”Dr. Ludwig Huber, esperto internazionale in materia di conformitàSicurezza negli ambienti regolamentatiQualificazione Operativa (OQ)Il processo di qualificazione operativa dimostra che lo strumento funzioneràsecondo le specifiche operative nell’ambiente previsto.Qualificazione delle prestazioni (PQ)Il laboratorio è responsabile della procedura di PQ che dimostra che il Bioanalizzatore 2100 sta funzionando secondo specifiche appropriate per l’uso di routine e produce dati affidabili,accurati e riproducibili.INSTALLAZIONEQualificazione dell’installazioneSTART UPQualificazione OperativaFUNZIONAMENTOQualificazione delleprestazioniIQ OQ PQPROGETTAZIONEQualificazione del designDQ7Specifiche dello strumentoSupporto a strumenti e applicazioniRiparazione in 24 ore, garanzia estesa, applicazione e servizi di conformitàServizi di assistenza per hardware e softwareTutti i componenti del Bioanalizzatore 2100 sono coperti da garanzia per un anno. Il Bioanalizzatore Agilent 2100 ècoperto dal servizio di riparazione premium che prevede la sostituzione dello strumento in 24 ore o la restituzione per la riparazione con prestito di uno strumento sostitutivo.Questo riduce significativamente i tempi di fermo macchina.Il servizio di garanzia premium può essere inoltre ulteriormente ampliato:•Estensione per uno o più anni della garanzia per tutti i principalicomponenti del Bioanalizzatore 2100,compresi PC, stampante e software,con servizio di “Express exchange”dello strumento. Inoltre, è compreso un intervento annuale dimanutenzione preventiva (PM).•Estensione per uno o più anni della garanzia “Express exchange” per il Bioanalizzatore 2100, con lapossibilità di includere il PC Agilent e la stampante.•Estensione per uno o più anni della garanzia del software, comprensiva di assistenza illimitata, aggiornamenti automatici e gratuiti del software e bollettini sullo stato del software.Supporto applicativoSono disponibili due tipi di assistenza applicativa on-site: •Servizi di start-up, per permettere ai nuovi utenti di acquisire familiaritàcon l’hardware, il software e l’applicazione scelta.•Servizi operativi, comprendenti la risoluzione dei problemiapplicativi e il corso sull’utilizzo del kit LabChip scelto. Il corso comprende l’esecuzione di analisi con campioni del cliente.Servizi di conformità per la qualificazione del sistemaPer gli attuali* sistemi con Bioanalizzatore 2100, Agilent offre servizi completi di qualificazione dell’installazione (IQ) e di qualificazione operativa (OQ). I servizi vengono forniti esclusivamente da tecnici Agilent appositamente addestrati e serie completa di servizi per la conformitàcomprende la qualificazione di esperimenti,cartucce e strumenti multipli.*Non disponibile per strumenti serie “A” venduti prima del 2001.Bioanalizzatore Agilent 2100Una piattaforma, possibilità infinite per l’analisi di DNA, RNA, proteine e cellulePer ulteriori informazioni: /chem/labonachip Acquista online:/chem/store Visita il nostro sito:/chem eMail:************************© Copyright 2008 Agilent Technologies Pubblicazione Febbrario 1, 2008Numero pubblicazione 5989-7725ITE。

衰减器及驱动器选择

衰减器
36 固定衰减器 (续)
Agilent 8490D
Agilent 8493A, B
2 Flats 180° apart 7 (0.275) across
8 (0.312)
7.6 13 8 (0.30) (0.50) (0.312)
A
Dimension A
5
3, 6, 10, 20 dB: 27 (1.06) 30, 40 dB: 29 (1.14)
125 W peak 500 W/ms max. 每一脉冲 (5.8 至 18 GHz)
3dB
6dB
0.3
0.3
衰减精度 (± dB)
10dB 20dB
30dB 40dB 50dB 60dB 连接器
0.5 0.5
1.0 1.5 1.5 2 N (m, f)
0.3
0.3, 至 12.4 GHz 0.6 0.6, 至12.4 GHz 1.0 1.5 1.5 2 N (m, f)
–0.03
–0.06
使用 2.4 mm 连接器, 该连接器有较大的接触表面, 能实现可
5
–0.09
靠和可重复的连接。对 3.5 mm 连接器的设计验证测试表明
–0.12
即使在1000 次连接后, 也不会造成测试的降级。对于步进衰减器来说, 内部射频连接的重复性也是要关心的问题。Agilent
–0.15
Agilent 11581A, 11582A, 11583C 成套衰减器
这是装在胡桃木盒中的一套四个 (3, 6, 10, 20dB) 衰减器。Agilent 11581A 是成套的 Agilent 8491A 衰减器; Agilent 11582A 是成套的 Agilent 8491B 衰减器; Agilent 11583C 是成套的Agilent 8493C衰减器。这些成套衰减器适用于校准实验室, 或是要求精确了解衰减值和 SWR 的场合。

玉米自交系响应高温、干旱胁迫的关键基因及通路

玉米自交系响应高温、干旱胁迫的关键基因及通路作者：姚启伦霍仕平张俊军来源：《江苏农业学报》2021年第01期摘要：以4个不同的玉米自交系为材料，对高温、干旱处理后的苗期植株进行转录组测序。

玉米自交系响应高温和干旱胁迫的差异表达基因（DEGs）分别为6 966和6 272个，在高温和干旱胁迫下4个玉米自交系相同的DEGs分别是705和871个。

同时响应高温和干旱的DEGs有100个。

在耐旱、耐热性强的玉米自交系中鉴定出18个特异的DEGs，其中锌指转录因子、WRKY转录因子、GT转录因子和B2热激转录因子在胁迫响应中发挥关键的调控作用。

KEGG通路分析结果表明，耐旱、耐热性强玉米自交响应高温干旱胁迫的DEGs富集在生物学过程、分子功能、代谢过程、遍在蛋白代谢和氮代谢途径5條通路。

热带、亚热带玉米种质的耐旱、耐热性强于温带玉米种质，可在热带、亚热带玉米种质中有效筛选耐旱、耐热基因。

关键词：玉米自交系;高温、干旱胁迫;转录组;差异表达基因;通路中图分类号： S513 文献标识码： A 文章编号： 1000-4440（2021）01-0029-09Key genes and pathways of maize inbred lines responding to heat and drought stressYAO Qi-lun1， HUO Shi-ping2， ZHANG Jun-jun3（1.School of Advanced Agriculture and Bioengineering， Yangtze Normal University，Chongqing 408100， China;2.Chongqing Three Gorges Acodemy of Agricultural Science，Chongqing 404100， China;3.Heilongjiang Hetian Fengze Agriculture Science and Technology Development Co.， Ltd.， Harbin 067500， China）Abstract： Transcriptome sequencing of the four different maize inbred lines subjected to high temperatures and water deficits， was performed at the stage of seedling. In total， 6 966 heat-responsive and 6 272 drought-responsive differentially expressed genes（DEGs） were identified in the four maize lines. In addition， 705 and 871 DEGs were identified as being commonly associated with heat and drought stress， respectively. There were 100 DEGs responding to both heat and drought stress. Eighteen DEGs were identified in maize inbred lines with strong drought tolerance and heat tolerance. The zinc finger transcription factor， WRKY transcription factor， GT transcription factor and B2 heat shock transcription factor played key regulatory roles in stress response. Analyses of KEGG pathway enrichment showed that the pathways such as biological process， molecular function， metabolic process， protein ubiquitination metabolism and nitrogen metabolism were the most highly enriched in the maize lines tolerant to heat and drought. It can be concluded that tropical and subtropical maize is tolerant to heat and drought， implying that it is plausible to screen goal genes in the tropical and subtropical germplasm.Key words： maize inbred lines;heat and drought stress;transcriptome;differentially expressed genes（DEGs）;pathways高温和干旱是抑制玉米生长发育、制约玉米产量的主要非生物胁迫因子[1]。

Agilent Technologies高灵敏度DNA套件和SureSelect靶向富集平台应用说明

AuthorsKirill Gromadski Ruediger Salowsky Susanne Glueck Agilent Technologies Waldbronn, Germany GenomicsImproving sample quality for target enrichment and next-gen sequencing with the Agilent High Sensitivity DNA Kit and the Agilent SureSelect Target Enrichment PlatformApplication NoteAbstractNext-generation sequencing (NGS) has revolutionized the genetic landscape.It is a lengthy, labor-intensive process that yields results never before achieved. As a result, it is imperative that the quality of the DNA sample be evaluatedfrom the start, as most NGS sample preparation protocols require PCR amplifica-tion to generate DNA libraries prior to sequencing. The likelihood of artifact gener-ation could contribute to bias, affecting the potential results. The High Sensitivity DNA Kit used with the Agilent 2100 Bioanalyzer has been optimized with improved levels of detection. The improved sensitivity allows the numbers of library PCR cycles to be reduced, removing amplification bias and significantly improving the quality of NGS data with increased accuracy.This Application Note describes how the Agilent 2100 Bioanalyzer High Sensitivity DNA Kit can be used to provide quantitative and qualitative information about the DNA samples used in the Agilent SureSelect Target Enrichment System.of the sheared genomic DNA, and to assess the quality and size distribution of the PCR amplified sequencing library DNA. After post-hybridization amplification, the Agilent 2100 Bioanalyzer can be used to determine the quality and the concentration of the PCR-amplified capture DNA before sequencing.This Application Note describes how the High Sensitivity DNA kit and the Agilent 2100 Bioanalyzer can be used before sequencing to reduce the num-ber of required PCR cycles. This reduces amplification bias, thus improving the quality of DNA libraries created during the SureSelect Target Enrichment workflow. ExperimentalDNA library preparationThe DNA library was prepared for Illumina’s Genome Analyzer II sequencers according to manufactur-er’s instructions.SureSelect Target EnrichmentThe SureSelect Target Enrichment for the Illumina single-end sequencing platform, consisting of three main steps; sample preparation, hybridiza-tion and post hybridization amplifica-tion, was carried out as described in the manual.316 DNA samples obtained after the post-hybridization amplification with different numbers of PCR cycles (4-18) were used for DNA analysis with the Agilent 2100 Bioanalyzer.High Sensitivity DNA analysis with the Agilent 2100 BioanalyzerThe on-chip DNA electrophoresis was performed on the Agilent 2100 Bioanalyzer in combination with the Agilent High Sensitivity DNA kit, according to the High Sensitivity DNA kit guide.4A dedicated High Sensitivity DNA assay is available with the Agilent 2100 Expert software (revision B.02.07 or higher). An integration region from 100 to 2000 bp wasused for all samples for smear quantification.DNA quantificationIn addition to the fluorescence-based DNA quantification on the Agilent 2100 Bioanalyzer, the Qubit fluorometer and the Qubit Quant-iT dsDNA BR Assay kit were used for DNA quantification according to the manufacturer’s instructions.IntroductionThe Agilent 2100 Bioanalyzer, an automated on-chip electrophoresis system,has already proven to be a valuable tool for automated sizing and quantification of various double-stranded DNA sample types relevant for the next-generation sequencing (NGS) sample preparation workflow.1 The Agilent 2100 Bioanalyzer with the DNA 1000 kit is recommended by NGS platform providers for measuring DNA sample quality prior to sequencing runs. These quality checks reduce time and resources wasted by low-quality samples. Recently, a High Sensitivity DNA kit was developed which offers improved sensitivity for checking the size and quantity of pre-cious low concentrated DNA starting material or DNA libraries down to a concentration of 100 pg/µL.Next-generation sequencing technolo-gy has brought high throughput to genome sequencing, but the new processes lack the ability to target specific areas of a genome. The SureSelect Target Enrichment System, enables genomic areas of interest to be sequenced exclusively. This cre-ates process efficiencies that reduce costs and allow more samples to be analyzed per study.2The Agilent High Sensitivity DNA Kit and the Agilent 2100 Bioanalyzer can be used for qual-ity control at several steps during the SureSelect Target Enrichment work-flow. During the sample preparation, the Agilent 2100 Bioanalyzer is used for quality control and sizing selectionResults and discussionThis Application Note describes how the Agilent High Sensitivity DNA kit and the Agilent 2100 Bioanalyzer can be used to further improve the quality of DNA sequencing libraries enriched by the SureSelect kit. For this purpose,16 amplified and purified DNA samples from the post-hybridization PCR ampli-fication step were analyzed with the High Sensitivity DNA kit and the 2100Bioanalyzer prior to sequencing on the Illumina platform.Figure 1 shows electropherograms of typical PCR amplified DNA libraries.The electropherograms show a typical smear from 150 to 350 nucleotides.The primers/primer-dimers migrated very close to the lower marker, but did not affect the analysis. The excellent sensitivity of the High Sensitivity DNA kit allowed the amplified DNA to be detected and reliably quantified,even after only four PCR cycles(figure 2). As expected, DNA concen-tration increased with the number of PCR cycles. Above 14 PCR cycles, the increase in DNA concentration was no longer linear and becomes saturated (figure 1B). When using 10 or more cycles, the DNA concentration was outside the quantitative range of the High Sensitivity assay. These samples were diluted with TE buffer in the indi-cated dilution ratios (figure 1B) prior to the analysis on the Agilent 2100Bioanalyzer.Figure 1PCR-amplified DNA library derived from the SureSelect Target Enrichment workflow, analyzed with the High Sensitivity DNA kit.(A) Overlay of DNA electropherograms obtained after 4 to 10 PCR cycles as well as TE buffer blank (black). The number of PCR cycles is indicated in the electropherogram overlay.(B) Overlay of DNA electropherograms obtained after 12 to 18 PCR cycles. The number of PCR cycles and the dilution ratios are indicated in the electropherogram overlay.B300350400450[FU]25020015010050035300350[FU]A2502001501005001002003004006001000200010380[bp]351002003004005006001000200010380[bp]10 cycles8 cycles6 cycles 4 cycles16 cycles (1:10 dil.)14 cycles (1:10 dil.)12 cycles (1:10 dil.)The key observation clearly shown in figure 1B, is that the quality of the PCR product depended on the number of PCR cycles performed. After 14 PCR cycles, an additional DNA smear at approximately 500 bp was detected in the electropherogram. This PCR arti-fact could potentially affect the effi-ciency of an NGS experiment. When running amplifications with PCR cycles below 14 this PCR artifact was not observed. DNA library analysis with the High Sensitivity DNA kit allows the number of required PCR cycles to be reduced. This results in fewer amplification-related artifacts, significantly improving the DNA sam-ple quality for downstream sequenc-ing. It should be expected that the improved DNA sample quality due to the reduced number of PCR cycles will positively affect the outcome of the downstream sequencing by reducing allelic bias, single-stranded DNA gen-eration, and duplicate sequences.5 The DNA concentrations determinedwith the High Sensitivity DNA kit were also compared with the DNA concen-trations measured with a fluorometer (table 1). The DNA samples obtained after 4 to 10 PCR cycles were mea-sured directly without dilution; all other samples were diluted as indicat-ed in figure 1. The selected assay on the fluorometer permitted DNA quan-tification only after 10 or more PCR cycles. The High Sensitivity DNA kit provided a reproducible DNA quantifi-cation after only four PCR cycles. For added confidence in our results, each DNA sample was measured four times on two different High Sensitivity DNA chips with the Agilent 2100 Bioanalyzer.Fluorometer2100 Bioanalyzer Deviation fromDNA conc. [ng/µL]DNA conc. [ng/µL]fluorometer [%]-0.090 ± 0.015--0.083 ± 0.007-6-0.304 ± 0.017--0.301 ± 0.010-8- 1.13 ± 0.07-- 1.34 ± 0.07-10 3.4 4.49 ± 0.1632.13.44.54 ± 0.1733.83.3 3.68 ± 0.2210.34.1 4.03 ± 0.23-1.01213.414.5 ± 0.58.012.513.9 ± 0.611.81436.439.8 ± 3.59.443.651.6 ± 3.318.31851.063.7 ± 3.424.961.470.0 ± 5.414.0Table 1Comparison of DNA quantification with a fluorometer and the Agilent 2100 Bioanalyzer. 16 different DNA samples obtained from the post-hybridization amplification step of the SureSelect Target Enrichment workflow were quantified with the fluorometer and the Agilent 2100 Bioanalyzer.The DNA samples were measured directly (4 to 10 PCR cycles) or after dilution (12 to 18 cycles)as indicated in figure 1. The standard deviation for the total DNA concentration measured with the Agilent 2100 Bioanalyzer was calculated from four data points measured on two different DNA chips.From To Corrected %of Average S izedistribution Concentration Molarity [bp]area total size [bp]in CV [%][pg/µL][pmol/L]2,000395.85125412.4283.331,713.8[p]35[FU]15010050100150Region 1Region 12003004005006001000200010380[bp]4 cyclesFigure 2Electropherogram obtained after 4 PCR cycles. The integration region from 100 to 2000 bp was used for smear quantification.Above 10 PCR cycles, overall DNA concentrations are generally similar for both methods. Variances in the DNA concentration determined with the fluorometer and the on-chip elec-trophoresis could be due to the differ-ences in technologies. Different fluo-rescent dyes are used, and the quan-tification by the Agilent 2100 Bioanalyzer is preceded by an elec-trophoretic separation of the sample. Additional variances were introduced by diluting the samples. Figure 3 graphically summarizes the data from table 1. The DNA concentra-tions obtained through both DNA quantification methods were plotted against the number of PCR cycles. Both methods, the fluorometer and the on-chip electrophoresis, clearly show the expected sigmoid amplification rate. The results obtained with both methods are in good agreement with each other. The Agilent 2100 Bioanalyzer provides DNA quantifica-tion as well as additional valuable information on the quality of the enriched DNA library.The insert in figure 3 shows the same data in double logarithmic scale to demonstrate the linearity of both methods. The linear dynamic range for the SureSelect DNA samples analyzed with the High Sensitivity DNA assay and the Agilent 2100 Bioanalyzer was determined to be 80 to 5000 pg/µL with r2= 0.9996. The last data point, after 18 PCR cycles, was not taken into account for this linearity analysis,as the PCR begins to saturate after 14 cycles.Figure 3Comparison of DNA quantification with a fluorometer and the Agilent 2100 Bioanalyzer. The obtained DNA concentrations (table 1) were plotted against the number of PCR cycles, Agilent 2100 Bioanalyzer ( black), fluorometer ( blue). The insert shows the same data in double logarithmic scale to demonstrate the linearity of both methods.References1.“Performance characteristics of the High Sensitivity DNA assay for the Agilent 2100 Bioanalyzer”, Agilent Technologies Technical Note, publica-tion number 5990-4417EN,2009.2.Gnirke, A., Melnikov, A., Maguire, J.,Rogov, P ., LeProust, E.M., Brokman,W., Fenell, T., Giannoukos, G., Fisher,S., Russ, C., Gabriel, S., Jaffe, D.B.,Lander, E.S. and Nusbaum, “Solution hybrid selection with ultra-long oli-gonucleotides for massively parallel targeted sequencing”, C., Nature Biotechnology, 27 (2), 182-189,2009.3.“SureSelect Target Enrichment System, Illumina Single-EndSequencing Platform Library Prep”,Agilent Technologies Manual,reference number G3360-90010, 2009.4.“Agilent High Sensitivity DNA KitGuide”, Agilent Technologies Manual,reference number G2938-90321, 2009.5.Quail, M.A., Kozarewa, I., Smith, F.,Scally, A., Stephens, P .J., Durbin, R.,Swerdlow, H., and Turner, D.J.;“A large genome center’s improve-ments to the Illumina sequencing system”; Nature Methods, 5 (12) 1005-10,2008.ConclusionQuality control of DNA samples after library generation derived from the SureSelect Target Enrichment work-flow can easily be performed with the High Sensitivity DNA kit and the Agilent 2100 Bioanalyzer.The High Sensitivity DNA kit offers increased sensitivity for DNA analysis down to pg/µL concentrations across a broad linear dynamic range. This enhanced performance allows the number of required PCR cycles to be significantly decreased, eliminating PCR artifacts, while still reliably quan-tifying the sample. The improved DNA quality will improve the outcome of downstream sequencing analysis,maximizing throughput efficiency while minimizing cost per sample Therefore, the number of required PCR cycles can be significantly decreased,eliminating PCR artifacts, while still reliably quantifying the sample./chem/DNA© Agilent Technologies, Inc., 2010, 2016 Published March 1, 2016Publication Number 5990-5008ENFor Research Use Only. Not for use in diagnostic procedures.The information contained within this document is subject to change without prior notice.。

Agilent 2100 Bioanalyzer

One platform –possibilities ArrayAgilent 2100 BioanalyzerOur measure is your success.products| applications | software | servicesAgilent 2100 bioanalyzer Applied lab-on-a-chip technologyThe Agilent 2100 bioanalyzer is the industry standard for RNA sample QC and has replaced labor-intensive gel electrophoresis for this application. It is also rapidly replacing gel electrophoresis for DNA fragment analysis and SDS-PAGE analysis of protein samples. A unique feature of the Agilent 2100 bioanalyzer is that it can be used for both electrophoretic separation and flow cytometric analysis of cell fluorescence parameters. This versatility makes the Agilent 2100 bioanalyzer an indispensable tool for the molecular biologist and biochemist.Miniaturization of analytical instrumentation has many advantages over conventional techniques. These advantages include improved data precision and reproducibility, short analysis times, minimal sample consumption, improved automation and integration of complex workflows. Launched in 1999, the Agilent 2100 bioanalyzer was the first commercially available instrument to use microfluidics technology for the analysis of biological samples.Choose from two configurations:•Agilent 2100 bioanalyzer with exchangeable cartridges for electrophoresis and flow cytometry applications •Agilent 2100 electrophoresis bioanalyzer with exchangeable cartridge for electrophoresis applications only Both instrument configurations include:1. Chip priming station2. Chip vortexer3. Agilent 2100 expert software for instrument control anddata analysis (expandable for full compliance support in regulated environments)4.Service and support on system and applications23•On-chip flow cytometryThe easy way to acquire dual-color, cell-based fluorescence data•RNA quality check with RINThe industry standard for RNA analysis offering total RNA, mRNA and Small RNA’s data including RIN algorithm (RNA Integrity Number)A wide range of applicationsA single compact system with ready-to-run kits•DNA size and quantitySmart, high resolution separation and quantification of DNA•SDS-PAGE replacement for Protein analysisThe fast and reliable way to determine the quantity and purity of proteins from Coomassie down to silver stain sensitivityThe Agilent 2100 bioanalyzer offers a broad range of pre-validated analysis kits combined with an easy-to-use benchtop system The Agilent bioanalyzer 2100: Automated, fast analysis with excellent data quality•Ready-to-use assays and pre-packaged reagent kits •Minimal sample consumption(1-4 µL) and results within 30 minutes•Replaceable electrode cartridge for contamination-free switch of methods•Improved assay accuracy and precision •Comparable results from lab to lab•Digital data for convenient analysis, archiving and storage •Various data-display options as gel view,electropherograms and tables•Easy to use with simplified sample comparison •Minimum exposure to hazardous materials •Supports compliance with 21 CFR Part 11BiochemicalAnalysisRNARNA Integrity RNA QuantificationSmall RNA SizingWhole CellsDNADNA Sizing DNA QuantificationProteinProtein Sizing Protein QuantificationProtein PurityEasy to use Simplified sample comparison •Ready-to-use reagent kits•Quick-start instructions•Chip preparation in less thanfive minutes•Minimal use of hazardouschemicals and waste disposal •Sample volumes in the µL rangeThe sample moves through themicrochannels from the samplewell.The sample is injected into theseparation channel.Sample components areelectrophoretically separated.Components are detected bytheir fluorescence and translatedinto gel-like images (bands) andelectropherograms (peaks).Principle of lab-on-a-chip operation1. 3. 4.2.4Get the most out of your digital dataPowerful software for the analysis of RNA, DNA, proteins and cellsThe Agilent 2100 expert software is the single solution specifically created for the bioanalyzer system. Built on the experience of several generations of bioanalyzer software it offers the flexibility of the integrated 2100 expert platform, with features carefully designed to help you get the most out of your digital data.Benefits at a glance•Powerful data evaluation tools – single platform for all available assays•Unique RNA Integrity Number (RIN) algorithm for unbiased total RNA integrity assessment•Ready for compliance – support system validation services (IQ and OQ) and 21 CFR Part 11 compliance using the optional security pack•Color-coded result flagging tool – an easy-to-use, rule-defined system for automated QC and data evaluation•Flexible results tables and graphics allow easy instrument control and support standard and advanced user modes•Fast single click overlay, scaling and zooming features allow quick comparison of up to 48 samples within one chip or across multiple chips•Multiple exportable data formats allow flexible data exchange•Free data-review software enables offline evaluation and sharing•Efficient integrated diagnostic tools minimize system downtime•Improved smear analysis for RNA, DNA and protein samples•Native XML file format for seamless data integration across platforms and projectsSetup and startAnalyze and compare Various graphicalviews, such as thegel-like formatshown here, aresupported by tablesof additionalinformation andfacilitate easyanalysis of results.Setup of samples,choice of methodand start of run isconvenientlyavailable form anintuitive userinterface givingaccess to 2100Instruments.Print reportsReports can beconfigured easilyby users to meettheir individualneeds. Color-codedflagging of resultsallows usersto see at a glancewhether specificresults arewithin limits.56Agilent supports your validation process from start to finishFor scientists working in regulated environments, the Agilent 2100 expert software supports software andhardware IQ and OQ/PV in a dedicated validation context.Design Qualification – DQ DQ defines the functional and operational specifications of the 2100 bioanalyzer system and ensures that it has all the necessary functions and performance criteria. Documents provided for the 2100 bioanalyzer system include:•Declaration of system validation •Declaration of conformity for instrument to manufacturing specificationAgilent software tools and services pave the way for complicanceBiopharmaceutical companies bear the extra costs and workload associated with quality control and regulatory compliance. For companies to achieve compliance, laboratory and production procedures have to be standardized and reproducible, while meeting good laboratory practice (GLP)and good manufacturing practice (GMP). Agilent helps to ensure consumer safety while minimizing the impact on biopharmaceutical companies with the Agilent 2100bioanalyzer for standardized, reliable quality control.Agilent supports the validation process with DQ documentation and IQ and OQ/PV tools and services. The security pack software for the Agilent 2100 bioanalyzer supports all 21 CFR Part 11 requirements. This includes the handling of electronic records, data security, data integrity and audit traceability. The Agilent 2100 expert software is a prerequisite for the Agilent 2100 expert security pack functionality.•Declaration of conformity for instrument according to ISO/IECGuide 22 and CEN/CENELEC EN 45014•Declaration of conformity for chips and reagentsInstallation Qualification – IQ IQ ensures that the Agilent2100 bioanalyzer instrument and the 2100 expert software are installed correctly upon delivery.“I believe Agilent provides the most complete range of compliance and validation services in the world. It offers global, multi-vendor,multi-product packages that let you make one call for virtually all your compliance needs.”Dr. Ludwig Huber,International compliance expertFeel safe in regulated environmentsOperational Qualification – OQ OQ is the process of demonstrating that an instrument will function according to its operational specifications in the selected environment.Performance Qualification – PQ Customers are responsible for the PQ which demonstrates that the 2100 bioanalyzer performs according to a specification appropriate for its routine use and produces reliable,consistent and accurate data.INSTALLInstallation QualificationSTART UPOperational QualificationOPERATEPerformance QualificationIQ OQ PQDESIGNDesign QualificationDQ7Instrument specificationsInstrument and application support24-hour repair, extended warranty, application and compliance servicesHardware and software support servicesAll Agilent 2100 bioanalyzer system components carry a one-year factory warranty. The Agilent 2100 bioanalyzer instrument is covered by the premium repair service of either 24-hourinstrument express exchange or return for repair with loan instrument. This significantly reduces the instrument downtime.The premium warranty service can be extended in the following ways to ensure maximum uptime.•One-year or two-year warranty extensions for all major Agilent 2100 bioanalyzer system components including, PC, printer, software and express exchange for the instrument.In addition, this warranty extension provides an annual system PM (Preventive Maintenance) service •One-year or two-year express exchange warranty extension for the Agilent 2100 bioanalyzer instrument, with the option of including the Agilent bundle PC and printer•One-year or two-year software warranty extension, comprising unlimited feature support,automated, free-of-charge software updates and software status bulletinsApplicationsupport servicesTwo on-site application related support services are available:•Start-up services to familiarize the new user with the hardware,software and an application of choice•Operational services including trouble-shooting for application-related problems and user training for a kit of your choice. Running customer samples is included in this trainingCompliance services for system qualificationFor all current Agilent 2100 bioanalyzer instrument bundles*, Agilent offers complete system (IQ) Installation Qualification and (OQ) Operational Qualification services. The Agilent 2100 bioanalyzer IQ and OQ services are exclusively provided by specially trained and certified Agilent service personnel. The complete suite of compliance services comprisesqualifying instrument and software for variable configurations.*Not available for "A" series instruments sold before 2001.Agilent 2100 bioanalyzerOne platform – endless possibilities for DNA, RNA, protein and cell analysisLearn more:/chem/labonachipBuy online:/chem/storeFind an Agilent customer center in your country: /chem/contactusU.S. and Canada1-800-227-9770*****************************Europe************************Asia Pacific****************************© Copyright 2008 Agilent Technologies Published February 1, 2008Publication Number 5989-7725EN。

安捷伦2100 生物分析仪简要操作说明

安捷伦2100 生物分析仪简要操作说明1．启动电脑，登陆刷卡系统。

2．开生物分析仪电源(位于后侧底部)，等待正面右上方状态灯显示绿色。

3．双击电脑桌面上“2100 Expert”图标，进入Instrument 主界面，确认COM Port 选择正确（COM选择1），左侧仪器示意图清晰显示(表示通讯正常) 。

4．按照试剂盒操作说明准备好注胶平台(注射器安装是否足够紧固以及注胶平台密封性是否完好，对于实验成功至关重要!)：5．按照试剂盒说明将2100 生物分析仪芯片槽的类型选择推杆推到正确位置(1 - 电泳，2 –细胞)。

6．按照试剂盒说明将芯片混匀仪转速设到正确位置(一般为2400rpm)。

7．取出试剂盒，平衡到室温约30 分钟，注意避光。

准备凝胶和染料的混合物，染料用完立即放回试剂盒避光。

(请参照相应试剂盒说明，注意离心过滤和混合的先后顺序以及离心转速的不同)8．取出9ul(核酸)或12ul(蛋白)凝胶染料混合物，加入电泳芯片的注胶孔(注意不要接触芯片底部，往芯片孔中加液时伸入底部不要靠壁!)9．将芯片放入注胶平台，拉动注射器推杆至1ml 刻度并扣紧上盖，压下推杆至固定架卡扣，计时(时间参考试剂盒说明)，到时间松开固定架卡扣，等待注射器推杆停止运动后将其缓慢拉回至1ml 刻度，松开注胶平台上盖。

10．从注胶平台取出芯片，并往右上方两个注胶孔各加入9ul(核酸)或12ul(蛋白)凝胶染料混合物。

(对于蛋白实验还要取12ul 去染色试剂至孔中)。

11．往电泳芯片上数字标记的样品孔及Ladder 孔中各加入5ul Marker。

(蛋白实验不需要加Marker 直接加6ul 样品或稀释的Ladder 并忽略下一步，对于RNA Pico 试剂盒还要往CS 孔中加入9ul Conditioning Solution)12．往Ladder 孔中加入1ul Ladder，数字标记的样品孔中各加入1ul 样品(RNA 样品和Ladder 以及蛋白样品的变性处理请参考相应试剂盒说明)。

Agilent 2100实验标准操作说明

Agilent 2100实验标准操作规程1.2. 准备凝胶染料混合液Gel-Dye Mix1.2.1. 取出Agilent DNA1000试剂盒中的DNA浓缩染料(蓝色)和DNA凝胶（红色），平衡试剂到室温。

大约30min。

1.2.2. 涡旋DNA浓缩染料（蓝色），然后用移液器吸取25ul染料，添加到一管DNA 凝胶中（红色）。

1.2.3. 充分涡旋混合液，离心。

转移到过滤柱中。

1.2.4. 2240g±20%，离心15min。

溶液避光保存在4°C。

1.3. 加载凝胶染料混合液Gel-Dye Mix1.3.1. 在实验操作前30min取出凝胶染料混合液室温平衡。

1.3.2. 取一块新的DNA芯片，放在芯片注胶平台上。

1.3.3. 吸取9ul凝胶染料混合液加到标记有G的孔内。

1.3.4. 确保注射器柱塞定位在1ml然后关闭芯片注胶平台。

1.3.5. 按压柱塞，直到被夹子卡住。

1.3.6. 等待计时60S，然后松开夹子。

1.3.7. 等待5S，然后拉回柱塞到1ml的位置。

1.3.8. 打开芯片注胶平台，吸取9ul凝胶染料混合液加到其他标记有G的孔内。

1.4. 加载标记物Markers吸取5ul Marker（绿色）加到12个样品孔内和Ladder孔内，不要让任何孔空着。

1.5. 加Ladder和加样1.5.1. 吸取1ul DNA Ladder（黄色）到标记有梯子的孔内。

1.5.2. 其他12个样品孔内，需要使用的孔内添加1ul样品，不需要使用的孔内，添加1ul去离子水。

1.5.3. 将芯片水平的放在芯片涡旋振荡器上，2400rpm涡旋1min（计时器计时）。

1.5.4. 5min之内芯片必须放在Agilent 2100上，按照软件提示进行DNA电泳操作。

1.6. 上机1.6.1. 打开电脑电源和仪器的电源。

1.6.2. 双击桌面上Agilent 2100 的软件图标，进入系统。

Agilent 2100生物分析仪

小的样品消耗量
• 无 RNA 酶污染 — 使用无 RNA 酶的试剂和芯片来避免分析过程中发生样品降解
• 洁净 — 最大限度地减少有害物质的接触，如溴化锭
Fluorescence
10
RIN = 9,2
8
6
4
2
0 19 24 29 34 39 44 49 54 59 64 69 Time [sec]
功能一键式操作，
轻链
• 洁净 — 最大程度地减少与染料或其它有害材料的接触
蛋白质 QA/QC • 监测蛋白降解和样品完整性 • 确定蛋白质污染物 • 在还原及非还原条件下分析蛋白
对照样
重链
稳定性试验
L对稳ad照定de样性r试验荧光
时间 (秒)
在还原条件下，用应激稳定性实验检测一个多克隆抗体的典型的降解和聚合方式。对照样品只表现与抗体轻链和重链相对应的两个峰。当备检样品处于 40 °C 下 12 周后，电泳图谱中出现了新的峰。Agilent 2100 生物分析仪可自动确定降解度。
• 微阵列或者 qPCR 实验前的 RNA 样品质量控制
• PCR/mPCR 和 RT-PCR 产物的 DNA 分析
• 蛋白表达分析
3. 样品组分通过电泳被分离
4. 各组分通过其荧光被检测并被转变成凝胶样图像（条带）和电泳图谱（峰）。
• 纯化过程中的蛋白纯度检测
• 在基因沉默或细胞凋亡研究中进行基于
培养 24h
荧光
百分率: 40.6
• 自动化 — 所需做的就是将 1 到 6 个样品加到一个芯片上即可开始分析。
• 芯片上染色步骤 — 加速工作流程。
荧光
总事件数: 1702
培养 24h

agilent2100原理

agilent2100原理Agilent 2100是一种高效的生物分析仪器，它基于电动毛细管电泳（CE）原理，可以用于DNA、RNA、蛋白质等生物大分子的分析。

本文将详细介绍Agilent 2100的工作原理及其在生物科学研究中的应用。

Agilent 2100采用了微流控芯片技术和毛细管电泳分离技术相结合的方式，使得分析速度更快、灵敏度更高，并且可以自动化进行样品分析。

它主要由高压供电系统、电泳芯片、检测器、电脑控制系统等组成。

在使用Agilent 2100进行分析之前，首先需要将待测样品进行标记。

常用的标记方法有荧光标记、放射性标记等。

标记完成后，样品通过注射器被输入到电泳芯片中，并在高压电场的作用下，被推动向电泳芯片的分离通道。

在电泳芯片中，分离通道通常是一条非常细且长的毛细管。

样品分子在电场力的作用下，按照其电荷和大小不同，以不同的速率向前迁移。

较小的分子会迁移得更快，而较大的分子迁移得较慢。

通过检测器可以实时监测样品分子的迁移状态，并生成电泳图谱。

Agilent 2100的检测器可以选择紫外检测器、荧光检测器等。

紫外检测器适用于检测无色分子，而荧光检测器适用于检测荧光标记的样品。

检测器会将检测到的信号传输给电脑控制系统，系统会自动分析信号并生成结果报告。

Agilent 2100在生物科学研究中有着广泛的应用。

例如，在基因测序领域，它可以用于检测DNA片段的长度和浓度，帮助研究人员进行基因序列的拼接和分析。

在蛋白质研究中，它可以用于检测蛋白质样品的纯度和分子量，帮助研究人员进行蛋白质结构和功能的研究。

除了在基础科学研究中的应用外，Agilent 2100还在临床医学、食品安全等领域发挥着重要作用。

例如，在临床医学中，它可以用于检测血液中的DNA或RNA，帮助医生进行基因诊断和个体化治疗。

在食品安全领域，它可以用于检测食品中的有害物质，保障食品质量和消费者的健康。

总结一下，Agilent 2100是一种基于电动毛细管电泳原理的生物分析仪器，通过微流控芯片技术和毛细管电泳分离技术的结合，实现了高效、快速、灵敏的样品分析。

20100601Agilent2100生物分析仪样本

2. 自动化启动芯片分析
3. 30 分钟内得到数字化数据实时观察数据显示
• 即时可用的试剂盒 • 快速启动指导说明 • 芯片准备不到 5 分钟 • 减少有害化学物质的使用和废物处理 • 样品体积低至微升级
• 一键式启动分析 • 预先内置的分析方法 • 系统采用内标计算结果 • 同时最多分析 12 个样品
Setup and start Analyze and compare
Print reports
5
Agilent 2100 专家软件
安全，符合法规认证的要求
安捷伦自始至终地支持您的认证过程
安捷伦 2100 专家软件为法规认证奠定基础
对于需要满足法规依从性的科学家，Agilent 2100 专家软件支持软件和硬件的安装认证、操作认证和性能认证。
• Agilent 2100 生物分析仪快速更换维修期可延长一年或两年，并可选择包括安捷伦捆绑的计算机
• 一年或两年软件授权期延长，包括各类功能支持、自动免费软件升级以及软件状态公告
应用支持服务
Agilent 2100 生物分析仪一个平台，无限可能 — 用于 DNA, RNA, 蛋白质和细胞分析
• 颜色标识工具为自动化结果显示和数据评估而设计，可设定标识规则且易于使用
• 灵活的图表模式提供简便的仪器控制，支持标准及高级用户模式
• 快速的一键点击实现叠加比对，缩放功能可让用户对一个芯片或多个芯片上的多达 48 个样品进行快速比较
• 多种可输出数据格式，允许灵活的数据交换
• 免费的数据分析软件能实现脱机数据评估及功能共享
“我认为安捷伦提供了世界上最完整的认证服务。包括全球多种供应渠道的各类产品的服务，一个电话就能满足您所有的认证需要”。 Ludwig Huber 博士，国际法规认证专家

微阵列比较基因组杂交技术与二代基因测序检测拷贝数变异的对比

微阵列比较基因组杂交技术与二代基因测序检测拷贝数变异的对比陈新周;王明珠;何丹;周慧;林方欣;李粉霞【摘要】目的对比分析微阵列比较基因组杂交技术(array-based comparative genomic hybridization,aCGH)和第二代基因测序技术(next generation sequencing,NGS)在基因组拷贝数变异(copy number variation,CNV)上的一致性,为临床检测CNV提供新的检测方法. 方法提取15例流产组织临床样本的基因组DNA,然后分别使用aCGH和NGS 2种方法对上述DNA进行检测,分析对比每个样本的CNV的数目. 结果 aCGH共检测到CNV数有109个,最小的片段有16 kb,最大的片段有34Mb,其中小于200 kb的有20个,大于200 kb小于1 Mb的有34个,大于1 Mb小于5 Mb的有26个,大于5 Mb小于34Mb的有29个.NGS共检出68个CNV,其中小于200 kb的有3个,检出率为15.0％;大于200 kb小于1 Mb的有22个,检出率为64.7％;大于1 Mb小于5 Mb的有20个,检出率为76.9％;大于5 Mb小于34 Mb的有29个,检出率为100.0％. 结论对于目前的数据量来说,NGS在检测大于5Mb的大片段CNV上检出率较高,与aCGH具有一定的一致性,可以应用于流产组织中CNV的检测.对于小片段的CNV的检测,需要增加相应的读取的数据量进行检测.【期刊名称】《分子诊断与治疗杂志》【年(卷),期】2016(008)006【总页数】4页(P385-388)【关键词】基因组拷贝数变异;第二代基因测序;微阵列比较基因组杂交技术【作者】陈新周;王明珠;何丹;周慧;林方欣;李粉霞【作者单位】广州市达瑞生物技术股份有限公司,广东,广州510665;南方医科大学检验与生物技术学院,广东,广州510515;广州市达瑞生物技术股份有限公司,广东,广州510665;广州市达瑞生物技术股份有限公司,广东,广州510665;广州市达瑞生物技术股份有限公司,广东,广州510665;南方医科大学检验与生物技术学院,广东,广州510515【正文语种】中文自然流产（spontaneous abortion，SA）在妊娠中的发生率为10％～15％［1］。

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One platform, endless possibilitiesThe Agilent 2100 Bioanalyzer is an indispensabletool for food chemists and biologists.Versatile, fast and mess-freeThe Agilent 2100 Bioanalyzer is the industry’s only platform that can cover your entire work-flow with a single compact system. The first commercial, analytical instrument based on lab-on-a-chip technology, the 2100 Bioanalyzer has proven to be an excellent alternative to antibody-based, labor-intensive gel electro-phoresis. This technology replaces subjective, time-consuming techniques associated with agarose or SDS-PAGE slab gels with fast, auto-mated, high quality digital data. Advantages of miniaturizationMiniaturization of analytical instrumentation has a number of advantages over conventional techniques:•Data precision and reproducibility•Short analysis times•Minimal sample consumption•Improved automation•Integration of complex workflows Answers within minutesThe 2100 Bioanalyzer provides you with a con-venient and productive way to gather and store experimental and routine test data. Automation and standardization of different processes on a chip give you high quality digital data fast, increasing lab productivity. You get answers within 30 minutes.Now you can have a multi-purpose platform that streamlines your workflows from product develop-ment to QA/QC of bulk materials and finished food products. The Agilent 2100 Bioanalyzer can handle all your needs, whether you want to measure protein content in seeds, bulk material or finished product; measure DNA or RNA for molecular detection of genetically modified organisms, allergenic species or pathogens; or count cells.Uniquely, the 2100 Bioanalyzer performs both electrophoretic separation and flow cytometric analysisof cell fluorescence parameters. It is rapidly replacing gel electrophoresis for DNA fragment analysisand SDS-PAGE analysis of protein samples.Pre-packaged LabChip kits, which includesample-specific reagents and chips, let youanalyze specific sample classes. A varietyof kits for RNA, DNA, protein and cell assaysare currently available to meet your needs.Why you need to test-drive the Agilent 2100 Bioanalyzer Advantages of the Agilent 2100Bioanalyzer•Faster than gels—digital data for up to12 samples within 30 minutes•Reproducible and complete digital data•Compliance Service options allow you to workwithin regulated environments (particularlyQA/QC labs)•2100 Expert Software for easy digital handlingand storage of all bioanalyzer data•Multiplex detection capabilities•Ready-to-use reagent kits•Quick-start instructions•Chip preparation in less than 5 minutes •Minimal use of hazardous chemicals and waste disposal•Sample volumes in the µL-range•Start analysis at the press of a button•Predefined protocols•System uses internal standards tocalculate results•Automated data analysis•Digital data can be filed in a databaseor shared•No user-dependent data interpretation Advantages of Agilent’s lab-on-a-chip technology•Minimal sample consumption and fast results•Improved assay accuracy and precision•Digital data for convenient archiving andstorage•Various data display options•Ease-of-use with simplified sample-to-samplecomparison•Minimum exposure to hazardous materialsDigital dataThe 2100 Bioanalyzer provides fully digital dataeasily shared with colleagues worldwide. Thefully functioning data analysis software is avail-able free at /cag/wad/registration/2100expert.asp2The electropherograms shown in Figure 1demonstrate multiplex detection of three nut species—Brazil nut (Bertholletia excelsa), pistachio nut (pistacia vera)and macadamia nut (macadamia integrifolia)—in a single run.Furthermore, the bioanalyzer, which uses only 1 µl of reaction mixture to load onto the LabChip,can reduce PCR volumes by up to 75 percent compared to standard gel-electrophoresis,which requires 10 to 15 µl, saving reagent costs.3Nuts and allergensThe 2100 Bioanalyzer is more cost effective and reduces labor compared to other test methods.Recent regulatory and policy changes in the United States (US), European Union (EU),Japan, Canada and Australia require labeling of multiple allergens in food products. EU regula-tions, for instance, identify eight nut species that must be declared on the ingredient list. Currently available allergen content tests for food products and materials often have difficulty in complex matrices and usually detect only a single allergen per test. In contrast, the 2100Bioanalyzer coupled with molecular detection techniques (PCR) can cost-effectively screen for multiple allergens using a single test.Figure 1: Cost-effective screening for multiplex PCR reaction product for nut allergen species in food using a single test.The 2100 Bioanalyzer’s gel-like image shows positive test results for pistachio nut (lane 4), Brazil nut (lane 5) and macadamia nut (lane 6), as well as for a mixture of these three nuts (lane 7) in wheat.Source: Campden and Chorleywood Food Research Association Group, Gloucestershire, UK.P is ta c h ioB ra zi lMa c a da mi aA dmi x tu r ePositive4Figure 2. Comparison of slab gel electrophoresis (A) with Agilent 2100 Bioanalyzer gel-like image (B) in detecting multiple GMO events in corn meal and soybean powder.Samples: L) molecular weight ladder (26, 34, 67, 89, 110, 147, 190, 242, 353, 404, 489 and 501 bp); 1) 0% MON810 corn;2) 0.1% MON810 corn; 3) 0.5% MON810 corn; 4) 1.0% MON810 corn; 5) 2.0% MON810 corn; 6) 5.0% MON810 corn; 7) commercial corn meal; 8) Allin positive control—0.5% Bt 176 maize and 0.5% Roundup Ready soybean; and 9) negative control (de-ionized water). Band identification: 118 bp—soy lectin; 153 bp—35S GMO; 217 bp—PCR reaction internal control; 278 bp—corn zein.Genetically modified organismsThe number of genetically modified organisms (GMO) and regulatory requirements for testing and labeling continue to increase. Many coun-tries now require GMO content labeling—or ban GMO materials altogether. Antibody tests to detect GMO proteins often require individual tests for each suspected GMO organism/event. Test accuracy can be problematic in processed materials where detected proteins may become denatured or damaged.The 2100 Bioanalyzer, in conjunction with an appropriate PCR kit, offers multiplex screening to detect and identify multiple GMO events in processed foods, improving test accuracy and reducing the number of tests, which in turn reduce costs. Figure 2 shows multiplex detection of GMO in corn meal and soya powder.AB35S GMO5Meat and fishThe multiplex detection capability of the 2100Bioanalyzer can also determine food product authenticity, its source and homogeneity. For example, meat authenticity is an important fac-tor for economic as well as religious and cultur-al reasons.Identifying authenticity and homogeneity of the meat source is difficult for processed meats in which proteins may be damaged by heat, ren-dering antibody-based tests inaccurate. Using molecular detection methods (PCR), the 2100Bioanalyzer successfully determines meat species and sample homogeneity in one test,reducing the number of tests required and thereby reducing labor and testing costs. Figure 3 shows the bioanalyzer’s ability to detect turkey, lamb or pork in a variety of meat samples while discriminating for other various matrices such as fish or grain.Figure 3. The 2100 Bioanalyzer can detect multiple targets at one time, including turkey, lamb or pork in a variety of meat samples. The dark band (lane 6) indicates a positive molecular test (PCR) for turkey.Negative tests (lanes 1 through 4) demonstrate test discrimination for other meat products (beef, chicken,lamb and pork).Figure 4.Agilent 2100 Bioanalyzer gel-like image showing PCR test designed to discriminate meat . Positive results are evident in lanes 1 through 5 for various meat products. Lanes 6 through 12 show negative results for grain and fish.Source: Campden and Chorleywood Food Research Association Group, Gloucestershire, UK.6Figure 5. Milk protein analysis. The 2100 Bioanalyzer protein assay provides rapid analysis of milk proteins. Shown here are two overlaid electropherograms of bovine milk (dil. 1:10; peaks identified by separately analyzing individual protein standards).Figure 6. Milk authenticity. The 2100 Bioanalyzer protein assay also provides a rapid means to deter-mine product authenticity quickly.Shown here are three overlaid electropherograms of milk samples (dil. 1:10) from different sources. In this example, milk samples from cow, buffalo and sheep are differ-entiated quickly based on relative milk protein content.Dairy: Milk proteinIn addition to DNA analysis for molecular detection applications, the 2100 Bioanalyzer can detect and quantify proteins to determine protein source, quality and content. Figure 5demonstrates determination of protein content to check the authenticity of the milk source (such as goat, sheep or cow) as well as deter-mine the protein content and quality for in-process or finished product QA/QC.Food safety applications253035404550(Sec)[FU][FU]Cow Buffalo Sheep300250200150100500253035404550(Sec)6005004003002001000Lower markerUpper markerSystem peaksβ−Lactoglobulinβ−Caseinα−Casein7WheatBecause the 2100 Bioanalyzer can electro-phoretically separate complex protein samples,providing both protein molecular weight and quantity, you can use pattern matching of the resulting electropherogram to determine prod-uct authenticity as an alternative (or comple-ment to) molecular detection methods. ForFigure 7. Protein patterns obtained by the 2100 Bioanalyzer’s protein chip identify various kinds of wheat. This figure covers extracts (1% SDS + 1% DTT)of six wheat varieties: Arrino, Tatiara, Rosella, Yanac, Sunbri and Sunsoft. The elution profiles from size-based capillary electrophoresis appear on the left,corresponding to the simulated gel patterns at right.Source: S. Uthayakumaran, I.L. Batey, C.W. Wrigley, Value Added Wheat CRC and Food Science Australia, North Ryde, Australia.example, verifying seed variety and quality is important economically because high quality and desirable seeds usually command apremium price. For many milled materials such as wheat flour, protein quality and quantity determine the suitability of the flour for the intended finished product. In a single test, thePattern matching can identify the particular strain of wheat,in this case Halberd.2100 Bioanalyzer can determine both thegenetic identity of various wheat varieties (DNA analysis) and protein content for either variety identification or product quality control. Figure 7shows wheat variety identification based on protein pattern.This information is subject to change without notice.© Agilent Technologies, Inc. 2007Printed in U.S.A. March 6, 20075989-6306ENFor more informationTo learn more about the Agilent 2100Bioanalyzer and additional food applications, go to:•/chem/labonachip •/chem/foods and click “Bioanalysis” and “Applications”You can also call 1-800-227-9770 (in the U.S.and Canada) or contact your local Agilent representative or Agilent Authorized Distributor.。