罗氏RNA提取说明书(05204909001_03.08)

RNA提取详细步骤及注意事项

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6、按1ml最初的Trizol加入0.5ml异丙醇，颠倒数次混匀，室温沉淀10分钟。 7、12,000g、 4℃离心10分钟，弃上清，在管底可见RNA沉淀（透明胶状）。 8、每毫升最初的Trizol加入1ml 75%乙醇（DEPC水配制），剧烈震荡。 9、 7,500g 、4℃离心5分钟，弃上清。再用离心机甩一下（>5,000rpm, 离心1分钟），小心吸尽液体。 10、待RNA略干后，加入20µl DEPC水溶解，-80℃冻存。
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磨样
2、主要提取流程
加 Trizol
去杂质
加氯仿
室温晾干溶解RNA
加乙醇
胶状物
测பைடு நூலகம்度
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保存
转移水相
3、详细步骤
1、剪取新鲜幼嫩叶片，置于液氮中慢慢反复研磨，直至其成为合格粉末为止。 2、加入1ml Trizol，晃动3-5下，再用枪吹打2-3下，确保全部裂解。 3、室温放置5分钟，使样品充分裂解。 12,000g、 4℃离心10分钟。 4、每毫升 Trizol加入0.2ml氯仿，猛烈晃动15秒，室温放置2-3分钟。 5、12,000g 4℃离心15分钟，然后吸取上层无色水相至一新的离心管中。
3、提取RNA整个过程中，尽量在低温下操作。
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4、在操作中当加入变性剂氯仿后，须剧烈震荡，这样才能彻底使两相混匀。
5、在提取核酸时，如样品浓度低，则应增加有机溶剂沉淀时间，－80℃下>30min或－20℃过夜将有助于增加核酸的沉淀量。
6、切勿让RNA过分干燥，否则将难溶解。
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谢谢老师的评阅！
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4、注意事项
1、所有离心管，枪头及相关溶液都必须无RNA酶污染。耐高温器物可放置于150℃烘烤4小时以去除RNA酶，其它器物去除RNA酶可考虑用0.01%的 DEPC水浸泡过夜，然后灭菌，烘干。溶液需用DEPC水配制。

罗氏RNA提取试剂盒使用指南说明书

Step 1: Sample Preparation & Nucleic Acid IsolationFor great results, use (click product names to learn more):Roche High Pure RNA Isolation KitRoche High Pure FFPET RNA Isolation KitRoche High Pure miRNA Isolation KitRoche RealTime ready Cell Lysis KitFrom which source (animal, organ, tissue) does the examined material originally come from? Which volume or mass or cell number was used for nucleic acid preparation?My MIQE Guide*Empowering results that matter Sponsored by Roche Applied Science Experiment title:Performed by:Date:Institution:Experimental design: How did you choose and set up your study (number of treated samplesand controls)Handling: Which tools or methods were used to obtain and process the primary samples (e.g., micro-dissection, macrodissection)?Method of processing and preservation: How was the sample treated and stored?If frozen – how and how quickly?If fixed – with what, and how quickly?If stored for longer: how and how long? (especially for FFPE samples)Extraction method:Which kit or instrument was used to extract/isolate the DNA/RNA from the starting material? Roche High Pure RNA Isolation Kit, High Pure FFPET RNA Isolation Kit, High Pure miRNA Isolation Kit, RealTime ready Cell Lysis Kit, or other (Please specify)Was the vendor’s protocol modified (If Yes, when, and how? e.g. by using additives)Did you do a DNAse or RNAse treatment? (If Yes, when?)Did you check for nucleic acid purity and integrity? If Yes: By using which instrument and method? What was the resulting purity (A260/A280)? What was the resulting yield? If No: Why not?Did you check for the presence of PCR inhibitors? If Yes: By using what (e.g. Cq dilutions, spike or other (please specify)If No: Why not?Final storage solution (e.g., buffer, H2O) for the purified total RNA:Storage time and temperature of the purified total RNA before use in RT-qPCR:Step 2:Reverse TranscriptionFor optimal results, use:Roche Transcriptor First Strand cDNA Synthesis KitRoche Transcriptor Universal cDNA MasterAmount of RNA and reaction volume:Priming oligonucleotide (if using gene specific primers) and concentration: Reaction temperature and time:Manufacturer of reverse transcription reagent(s) and catalogue number(s): Reverse transcriptase type and used concentration:Storage conditions of cDNA:Step 3:PCR Amplification and AnalysisFor best results, use:LightCycler® 480 Probes MasterFastStart Essential DNA Probes MasterFastStart Universal Probe Master (Rox)Target sequence and amplicon information: Target gene database sequence accession number:Location of amplicon:Amplicon length:Result of in silico specificity screen (BLAST, etc.):Information on pseudogenes, retropseudogenes or other homologs: Secondary structure analysis of amplicon:Determined by which method?Location of each primer relative to exons or introns (if applicable): Targeted splice variants:RTPrimerDB Identification Numbers: Manufacturer of oligonucleotides: Purification method:For probe-based assays: Probe type:qPCR reaction conditionsReaction volume and amount of cDNA/DNA per reaction: Primer, (probe), Mg2+ and dNTP concentrations: Polymerase identity:Buffer/kit manufacturer and identity (e.g., catalog number)Manufacturer and catalog number of plates or tubes and catalog number:Complete thermocycling parameters:Reaction setup: Was it manual or robotic? If robotic: Using which robot?Equipment: Which Real-Time PCR instrument was used? (Which Roche LightCycler® System or other (please specify)?)Validation of qPCR runs:Are you running a multiplex assay? If yes, please describe efficiency and limit of detection foreach assay:How did you check for specificity of amplification for each target (e.g., on a gel, by sequencing, melt-ing curve analysis or digest):For SYBR Green I assays: Cq of the non-template control reaction:Standard curve characteristics (slope and y-intercept):How many replicates did you use to establish the standard curve?(xx replicates per standard concentration)What was the lower and the upper limit of the standard curve?PCR efficiency calculated from slope:Confidence interval for PCR efficiency or standard error:r2 of standard curve:Information on linear dynamic range:Cq variation at lower limit: Confidence intervals throughout range:Evidence for limit of detection:How many reactions per run were used for controls? (please specify positive and negative controls, controls without template and No RT controls, e.g. Positive controls: 3 reactions in 5 replicates per 96 well plate)Data analysis:Vendor software: Which software type, version and algorithm provided by the PCR machine supplier was used to analyze the data?Specialist software: Which (if any) additional software was used? Self-developed algorithms,or other (please specify)Normalisation: Which reference gene(s) were used to calculate the relative expression of the studied genes?What was the reason for choosing these particular genes?Which algorithm (e.g., geNorm, bestkeeper, normfinder) was used to normalize for reference gene(s)Which principle was used for Cq calling?What was the number and of biological replicates used?How was their concordance?How many technical replicates were used, and at which step (RT or qPCR)? What was the observed repeatability (intra-assay variation)?What was the observed reproducibility (inter-assay variation, %CV)The MIQE guidelines empower results that truly matter. And so does Roche.Visit to discover all the materials you need for truly remarkable research results.* modified based on the list in the original MIQE guidelines publication with permission of the MIQE authors.For life science research only. Not for use in diagnostic procedures. LIGHTCYCLER and FASTSTART are trademarks of Roche.All other product names and trademarks are the property of their respective owners. NOTICE: This product may be subject to certain use restrictions. Before using this product, please refer to the Online Technical Support page () and search under the product number or the product name, whether this product is subject to a license disclaimer containing use restrictions.Published byRoche Diagnostics GmbH Sandhofer Straße 116 68305 Mannheim Germany© 2013 Roche Diagnostics. All rights reserved.*********** 1012。

OMEGA RNA提取步骤

OMEGA RNA 提取试剂盒步骤
1、将研磨好的冷冻植物组织100mg加入到离心管中，加入500ul Buffer RCL(使用前已加入β-巯基乙醇)，大力涡旋以使组织块状分散，均匀反应。

2、55℃孵育3min，室温下14,000×g离心5min
3、吸取上清液至gDNA过滤吸附住中，室温下14,000×g离心2min
4、丢弃gDNA过滤吸附柱，在收集管中加入等体积的BufferRCB，吹打混匀5-10次
5、吸取步骤4中混合液的1/2到HiBind RNA小吸附柱中，室温下10,000×g 离心1min，倒掉收集管中的液体
6、将步骤4中剩余的混合液再次加入到HiBind RNA小吸附柱中，室温下10,000×g离心1min，倒掉收集管中的液体
7、加入400ul RWC Wash Buffer，室温下10,000×g离心1min，扔掉收集管
8、将吸附柱放入新的2ml收集管中，加500ul RNA Wash BufferⅡ，室温下10,000×g离心1min
9、重复步骤8，再次用500ul RNA Wash BufferⅡ洗涤吸附柱，室温下10,000×g离心1min，倒掉收集管中液体，然后吸附柱空离，10,000×g离心2min，以使干燥
10、RNA溶解：将吸附柱放入新1.5ml离心管中，加30-50ul DEPC水，室温孵育2min,10,000×g离心1min(注：如果提取的RNA含量＞30ug，必须再次加入适量DEPC水溶解)。

自己翻译的罗氏tunel检测细胞凋亡试剂盒说明书

罗氏tunel检测细胞凋亡试剂盒说明书注意：Label溶液含有甲次砷酸盐和二氯化钴，严禁吸入和食入。

反应悬浮物收集于密闭、不易碎、有明确标识的容器中，按有毒废物处理。

除上表所列试剂外，还需准备以下溶液。

下表列出每步所需物品概览：特异性：TUNEL反应优先标记凋亡产生的DNA链断裂，从而辨别凋亡与坏死、以及由抑制细胞生长的药物或放射线产生的primary DNA链断裂实验干扰：假阴性：在某些型式的凋亡细胞中DNA链断裂可能缺失或不完全。

空间位阻，如细胞外元件可能阻止TdT到达DNA断裂处。

两种情况均能产生假阴性。

假阳性：在坏死晚期，可能产生大量的DNA片段DNA链断裂也可能在具有高增殖和代谢活动的细胞中出现。

两种情况均能产生假阳性。

为确认细胞死亡的凋亡型式，应认真进行每种细胞的形态学检查凋亡过程中产生的形态学改变尤其特征形式，因此，对于可以结果进行解释时，细胞形态评估是一项重要的参数样本：细胞离心涂片和细胞涂片在chamber slides上培养的黏附细胞冰冻或福尔马林固定、石蜡包埋样本分析时间：2-3小时，除外培养、固定和渗透检测次数：一个试剂盒50T试剂盒存储/稳定性：未开封试剂盒储存于-15～-25℃可稳定至标签上标明的效期。

1 流程图：2 样品准备2.1 黏附细胞、细胞涂片和细胞离心涂片需准备的其他试剂：Washing buffer：磷酸盐缓冲液（PBS）Blocking buffer封闭溶液：甲醇稀释的3% H2O2Fixation solution固定溶液：PBS配制的4%多聚甲醛，ph 7.4，新鲜配制Permeabilisation solution 渗透液：0.1%Triton1）X-100溶于0.1%柠檬酸钠溶液中，新鲜配制步骤：下表描述了细胞固定、内源性过氧化物酶封闭和细胞渗透过程。

2.2 组织部分2.2.1 福尔马林-包埋组织福尔马林包埋组织的预处理：可按4种不同的方式预处理。

RNA提取标准操作规程

研究机构名称：中国药科大学药代动力学重点实验室SOP编号：SOP名称：RNA提取标准操作规程1.目的本SOP的目的是为了保证RNA提取实验的标准化和可重复性。

2.前言本SOP涉及细胞及组织样品的RNA提取的标准化操作过程。

3.范围本SOP适用于中国药科大学药代动力学重点实验室的全体实验操作人员。

4.职责4.1细胞室PI应指派专人负责RNA提取实验前的培训。

4.2所有实验操作者应经由专人培训，明确RNA提取实验的原理及步骤后方可独立操作。

5.操作步骤5.1实验前准备RNA制备的关键是要抑制细胞中的RNA分解酶和防止所用器具及试剂中的RNA分解酶的污染。

因此，在实验中必须采取以下措施：戴一次性干净手套；使用RNA操作专用实验台：在操作过程中避免讲话等以防止实验者的汗液、唾液中的RNA分解酶污染。

5.1.1器具准备RNA实验用的器具建议专门使用，不要用于其它实验。

尽量使用一次性塑料器皿，若用玻璃器皿，应在使用前按下列方法进行处理。

（1）用0.1% DEPC （焦碳酸二乙酯）水溶液在37℃下处理12小时。

（2）然后在120℃下高压灭菌30分钟以除去残留的DEPC。

5.1.2试剂配制：（1）RNAiso Plus 试剂购于TaKaRa，货号：D9108A（2）氯仿（3）异丙醇（4）75%乙醇（DEPC处理水配制）（5）RNase-free水（制备方法：使用RNase-free的玻璃瓶，向超纯水中加入DEPC至终浓度01%（/0，过夜搅拌后，高温高压灭菌。

）5.2实验操作5.2.1RNAiso Plu的使用量参考RNAiso Plus使用量（ml）样品量10 cm2的贴壁培养细胞107的悬浮培养细胞1〜2100 m的白细胞2100 M全血250〜100 mg的普通组织样品150〜100 mg的特殊组织样品（肝、脾、骨及软骨等）215〜30 mg的植物材料（多糖和多酚含量不高的）15.2.2实验样品的研磨和匀浆。

rna提取具体操作流程

rna提取具体操作流程下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。

文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor. I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!RNA 提取是分子生物学实验中常用的技术之一，用于从细胞或组织中分离出 RNA。

RNA提取trizol试剂盒说明书

注：各种样品的最大使用量（1ml Trizol）操作步骤：1. 在液氮中将组织(单头飞虱)研磨成粉末，趁液氮尚未挥发光时，将粉末转移到1.5ml离心管中。

细胞经计数后直接加入离心管，然后5000rpm室温离心去上清。

每100mg组织或5×106个细胞加1ml的Trizol。

注意：如果组织量（1-10mg）或细胞数很少（1×102-1×104），在样品中加入800μl的Trizol，用枪头反复抽吸混匀，再加入糖原（终浓度为250μg/ml），剧烈振荡或用匀浆器匀浆。

2. 用1ml针筒，26-G号（6#）针头抽吸匀浆液两次以剪切基因组DNA，然后直接从针筒中将样品转移到无菌1.5-ml离心管中。

3. 加入200μl氯仿/异戊醇（24:1）或氯仿，剧烈振荡混匀30秒。

4. 台式离心机上，12000rpm，室温离心5分钟。

5. 将上清液小心转移到RNase-free 1.5ml 离心管中，加入等体积的异丙醇，室温下放置5分钟。

（注意：不要吸取任何中间层物质，否则会出现染色体DNA污染）6. 台式离心机上，12000rpm，室温离心5分钟。

7．小心移去上清液，防止RNA沉淀丢失。

8. 用70%酒精洗涤两次，每次700μl，12000rpm，室温离心2分钟。

9. 尽可能彻底地吸走上清，防止RNA沉淀丢失。

10. 真空离心干燥3-5分钟，或放在室温下使酒精完全挥发。

11. 沉淀用30-50μl DEPC-H2O溶解。

如发现沉淀难溶，68℃处理10分钟。

对于胰腺，肾等组织中RNase含量很高，沉淀用100%去离子甲酰胺溶解。

12．DNA的分析和定量：（1）测定样品在260nm和280nm的吸收值确定RNA的质量。

按1 OD=40μg RNA计算RNAD的产率：OD260/280在1.8-2.0视为抽提的RNA纯度很高。

若需精确量化，只有浓度在4μg/ml以上的样品适于用光度计测定。

RNA提取的操作步骤--经验

RNA提取的操作步骤准备试剂：氯仿，异丙醇，75℅乙醇，无RNase的水或0.5℅SDS(溶液均需用DEPC 处理过的水配制)操作步骤:1. 匀浆处理:a.组织将组织在液氮中磨碎,每50-100mg组织加入1ml TRIzol,用匀浆仪进行匀浆处理。

样品体积不应超过TRIzol体积10℅。

b.单层培养细胞直接在培养板中加入TRIzol裂解细胞，每10cm2面积(即3.5cm 直径的培养板)加1ml，用移液器吸打几次。

TRIzol的用量应根据培养板面积而定，不取决于细胞数。

TRIzol加量不足可能导致提取的RNA有DNA污染。

c．细胞悬液离心收集细胞，每5-10×106动物、植物、酵母细胞或1×107细菌细胞加入1ml TRIzol，反复吸打。

加TRIzol之前不要洗涤细胞以免mRNA降解。

一些酵母和细菌细胞需用匀浆仪处理。

2．将匀浆样品在室温（15-30℃）放置5分钟，使核酸蛋白复合物完全分离。

3．可选步骤：如样品中含有较多蛋白质，脂肪，多糖或胞外物质（肌肉，植物结节部分等）可于2-8℃10000×g离心10分钟，取上清。

离心得到的沉淀中包括细胞外膜，多糖，高分子量DNA，上清中含有RNA。

处理脂肪组织时，上层有大量油脂应去除。

取澄清的匀浆液进行下一步操作。

5. 每使用1ml TRIzol加入0.2ml氯仿，剧烈振荡15秒，室温放置3分钟。

6. 2-8℃10000×g离心15分钟。

样品分为三层：底层为黄色有机相，上层为无色水相和一个中间层。

RNA主要在水相中，水相体积约为所用TRIzol试剂的60℅。

7. 把水相转移到新管中，如要分离DNA和蛋白质可保留有机相，进一步操作见后。

用异丙醇沉淀水相中的RNA。

每使用1ml TRIzol加入0.5ml异丙醇，室温放置10分钟。

8. 2-8℃10000×g离心10分钟，离心前看不出RNA沉淀，离心后在管侧和管底出现胶状沉淀。

RNA 提取方法(TRIzol法)

RNA 提取方法（TRIzol法）一、试剂准备1、 TROzlo试剂、氯仿、75%乙醇（0.1% DEPC配制）。

2、塑料器皿需用0.1% DEPC水浸泡。

3、 0.1%DEPC水：100ml dd水中加入DEPC0.1ml，充分振荡，37℃孵育12h以上，121℃高压灭菌20min，于4℃保存。

二、操作步骤1、样品处理组织：50-100mg组织中加入1ml TROzlo试剂。

单层细胞：加入TROzlo试剂1ml/cm2平板。

悬浮细胞：处理前洗涤细胞，以防止RNA降解。

每5-10×105动物、植物或酵母细胞，或1×107细菌加入1 ml TROzlo试剂。

2、将上述样品于15-30℃静置5min，使核蛋白充分解离。

3、加入0.2ml（1 ml TROzlo试剂）氯仿，盖紧盖子，充分剧烈振荡15s并于15-30℃静置2-3min。

4、于2-8℃ 12000g离心15min。

离心后样品分层，上层水相中含RNA，下层有机相中含蛋白和DNA。

5、取上清，加入0.5ml异丙醇，轻轻混匀，于15-30℃静置10min后，在管底会出现胶状沉淀，即为RNA。

6、于2-8℃ 12000g离心10min后弃去上清。

7、向沉淀中加入1ml 75%乙醇，轻轻混匀。

8、于2-8℃ 7500g离心5min后弃上清9、将RNA样品凉干（不要彻底干燥），加入适量DEPC水溶解（可于55-60℃促溶10min）。

三、注意问题1、在加入氯仿之前（第1步），样品能于-60- -70℃保存至少一个月。

2、 RNA沉淀（第6步）在75%乙醇中于2-8℃能保存至少一周，于-5- -20℃能保存至少一年。

四、RNA定量RNA（mg/mL）=40×OD260×稀释倍数（n）/1000RNA纯品OD260/OD280=2.0五、RNA电泳1、用1×TAE电泳缓冲液制作琼脂糖凝胶，加1×TAE电泳缓冲液至液面覆盖凝胶。

RNA提取实验操作步骤、注意事项及问题指南

Trizol法RNA提取实验操作步骤、注意事项及问题指南（破碎组织→分离RNA→沉淀RNA→洗涤RNA→融解RNA→保存RNA）1 试剂：三氯甲烷（氯仿），异丙醇，75℅乙醇，无RNase的水、Trizol reagent2 材料与仪器：EP管匀浆器移液枪漩涡振荡器低温高速冷冻离心机冰袋电泳仪琼脂糖凝胶超净工作台3 操作步骤：3.1 准备工作A 玻璃匀浆器、75℅乙醇预冷，离心机预冷至4℃打开超净工作台紫外灯15分钟以上B 用酒精擦拭台面EP管标记3.2. 匀浆处理1 取一定量的纯化过的孢子悬浮液，12000rpm ，离心5min，弃上清a 或者用匀浆仪进行匀浆处理，悬浮液不经离心，直接加0.5ml Trizol reagent于冰上充分匀浆悬浮液样品体积一般不要超过Trizol体积的10%.，然后再加入0.5ml Trizol 冲洗匀浆器，转移至1.5ml EP管中b 直接在悬浮液中加入Trizol裂解细胞，加1ml Trizol.用取样器吹打几次.（离心取细胞，每5-10×106动物、植物和酵母细胞或每107细菌细胞加1ml Trizol。

加Trizol前不要洗涤细胞，以免降解mRNA。

一些酵母和细菌细胞可能需要匀浆仪处理）2 1ml Trizol Reagent，震荡混匀3. 将匀浆样品在15-30℃放置5mim，使得核酸蛋白复合物完全分离。

4. 可选步骤:4 ℃12 000rpm离心10min，取上清。

如果样品中含有较多蛋白、脂肪、多糖或肌肉、植物结节部分等，可离心去除。

离心得到的沉淀中包括细胞外膜、多糖、高分子量DNA，上清中含有RNA。

处理脂肪组织样品时，上层是大量油脂，应除去。

取澄清的匀浆溶液进行下一步操作。

5. 每使用1ml Trizol加0.2ml氯仿，盖好管盖，在漩涡振荡器上震荡15s，室温放置3min。

如不能涡旋混匀，可手动颠倒混匀2min代替。

6. 4 ℃12 000rpm，离心10-15min，样品会分成三层：红色的有机相，中间层和上层无色的水相，RNA主要在水相中，把水相（约600ul，约为所用Trizol试剂的60%）转移到新管中。

Omega公司RNA提取试剂盒说明书

RNA-Solv Reagent®RNA Isolation SolventWARNING: This reagent is toxic if swallowed and in contact with skin. Causes burns. After contact with skin, wash immediately with copious amounts of mild detergent and water. If you feel sick, seek medical advice at once and Quote UN2821.Product No:R6830-00 (5 ml)R6830-01 (100 ml)R6830-02 (200 ml)Storage Conditions:RNA-Solv is stable for at least 24 months®when stored at 2°C-8°C and yields reproducible results.IntroductionRNA-Solv® Reagent is a reagent system for the isolation of total RNA from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step RNA isolation method developed by Chomczynski and Sacchi (1).The sample is homogenized and lysed in RNA-Solv® Reagent which maintains the integrity of the RNA, while disrupting and denaturing endogenous RNases and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. RNA remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X10), and large quantities of tissue ( up to1 g) and cells 6(<10 ), of human, animal, plant, or bacterial origin. The simplicity 8of the RNA-Solv® Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA prepared in this manner can be used for Northern blot analysis, dot blot hybridization, poly(A) + selection, in vitro translation, RNase protection assay, and molecular cloning. For use in amplification by thermal cycling, treatment of the isolated RNA with RNase-free DNase I is recommended when the two amplimers lie within a single exon.Supplied By User•Chloroform (no isoamyl alcohol added)•Isopropyl alcohol•80% Ethanol (in DEPC-treated water)•RNase-free water•Tabletop centrifuge capable of 12,000 x g at room temperatureGeneral Notes Regarding RNase ContaminationWhenever working with RNA :•Always wear disposable gloves and change gloves frequently.•Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases.•In the presence of RNA-Solv® Reagent, RNA is protected from RNase contamination. Downstream sample handling requires that nondisposable glassware or plasticware be RNase-free.•Use only DECP-treated buffers. Add DEPC to a final concentration of 0.1%, incubate at 37C for 2 hours, andoautoclave at 121C. Do not add DEPC to Tris buffers. Suchobuffers must be prepared by using DECP-water.PrecautionUse only disposable polypropylene tubes for small samples and glass Corex tubes for larger samples. All tubes must be able to withstand 12,000 x g . Polystyrene tubes may crack with chloroform Before StartingA.Small Samples :To isolate RNA from very small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) of samples in 0.8 mL of RNA-Solv®, and add 1 mg RNase-free glycogen or yeast tRNA as carrier. This will improve yields obtained with precipitation.B.Difficult Animal Samples: Specimens containing large amounts of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and sperm, will require the following modification. After lysis/homogenization in RNA-Solv® Reagent, centrifuge at 12,000 x g for 10 minutes at room temperature to remove insoluble debris. Often a precipitate forms at the bottom of the tube, but with fatty tissue, a lipid layer will also form above the aqueous phase. The supernatant will contain the RNA and must be carefully transferred to a fresh 1.5 ml microfuge tube before proceeding.C. Interruption the procedure:Following lysis in RNA-Solv®Reagent and before addition of chloroform, samples can be stored at -70C for up to 3 months. In addition, once the RNA is oprecipitated in isopropanol, the pellet may be stored at -20C or -o70C for up to 1 year.oRNA-Solv Protocol for Total RNA Isolation®CAUTION: When working with RNA-Solv® Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20C-25C).o o1. Homogenization and lysis of samples: follow either method belowa) Tissue SamplesHomogenize tissue samples in 1 mL of RNA-Solv® Reagent per 50-100 mg of tissue using an appropriate mechanical homogenizer. Alternatively one can pulverize tissue in liquid nitrogen with mortar and pestle and transfer the powder to a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (Eppendorf, Cat No. 0030 120.973; VWR, Cat No. KT 749520-0000). The sample volume should not exceed 10% of the volume of RNA-Solv® Reagent used.b) Cells Grown in SuspensionPellet cells by centrifugation. Lyse cells in RNA-Solv® Reagent by repetitive pipetting. Use 1 mL of the reagent per 5-10 x 10of6 animal, plant or yeast cells, or per 1 x 10 bacterial cells. Washing8cells before addition of RNA-Solv® Reagent should be avoided as this increases the possibility of mRNA degradation and RNase contamination. For plant, fungal, and yeast cells mechanical or enzymatic homogenization may be required. Also, for plant, fungal, and yeast cells, we recommend the use of the E.Z.N.A.® Plant (R6627),Fungal (R6640), and Yeast (R6670) RNA Kits from Omega Bio-tek.c) Cells Grown in MonolayerLyse cells directly in a culture dish by adding 1 mL of RNA-Solv®Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a blue pipette tip. The amount of RNA-Solv®Reagent added is based on the area of the culture dish (~1 mL per 10 cm ). An insufficient amount of RNA-Solv® Reagent may result2in contamination of the isolated RNA with DNA. Always use more RNA-Solv® Reagent if in the lysate is too viscous to aspirate with a pipette.2. Add 0.2 mL of chloroform per 1 mL of RNA-Solv® Reagent. Cap sample tubes securely and vortex vigorously for 15 seconds. Incubate on ice for 10 minutes. This step is critical - do not change it.3. Centrifuge the samples at no more than 12,000 x g for 15 minutes 4E C.The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA remains entirely in the aqueous phase.4. Precipitation of RNA.Transfer no more than 80% of the aqueous phase to a fresh tube, and discard the lower organic phase. Precipitate the RNA from the aqueous phase by adding 500ìl of isopropyl alcohol per 1 mL of RNA-Solv® Reagent used for the initial homogenization. Incubate samples at room temperature 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes also at room temperature.Carbohydrate-rich samples: Plant samples of high polysaccharide content or animal tissues rich in glycosaminoglycans (proteoglycans) require the following modified precipitation method for obtaining pure RNA. Prepare Buffer A ( 1.2 M sodium chloride, 800 mM sodium citrate). Following step 3, add to the aqueous phase 0.3 ml isopropanol followed by 0.3 ml Buffer A per 1 ml RNA Solv ® Reagent used in step 1. Vortex to mix and centrifuge at no more than 12,000 x g for 10 minutes at room temperature. This high salt precipitation will reduce co-purification of complex carbohydrates.5. Wash RNA pellet. Discard the supernatant and wash the RNA pellet once with 1 ml 80% ethanol. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at room temperature.6.Reconstitute RNA. Carefully aspirate and discard the ethanol and briefly AIR DRY the RNA pellet for 2-5 minutes at room temperature. Do not use centrifugal devices equipped with a vacuum source as over-drying will lead to difficulty in re-dissolving RNA in water. Dissolve RNA in RNase-free water - a 5 minute incubation at 60 °C may be required. RNA can also be reconstituted in 100% formamide (deionized) and stored at -70°C.RNA is now suitable for RNase protection, northern analysis and reverse transcriptase reactions. For isolation of poly(A)+ RNA an additional ethanol precipitation is required. Add 1/8 X volume of RNase-free 3M NaAc, pH 6.0 followed by 2.5 X volume absolute ethanol. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge at 12,000 x g for 10 min at room temperature and discard the supernatant. Wash the pellet as before and reconstitute in DECP-treated water.Determination of Yield and QualityUV spectrophotometric analysis of the purified RNA is required for obtaining yield. To do so, dilute the RNA in an appropriate volume of TE buffer, pH 8.0 (not water; RNA yields low Abs ratio values if dissolved in acidic buffers) and measure absorbance at 260 nm and at 280 nm. RNA Conc = 40 ìg/ml X Dilution factor X Abs 260 nmTypical Abs 260 nm/ 280 nm ratios of 1.7-1.9 are obtained with the protocol. Yields vary depending of type and amount of starting material, and on condition of storage prior to processing. For assessing the quality of RNA, we recommend you perform denaturing agarose gel electrophoresis to confirm the integrity of purified material. Invariably, the full spectrum of RNAs, including 4S and 5S species are purified with RNA-Solv® Reagent.Expected Yields per 1 mg tissue or 10 cells:6Liver and spleen, 5-10 ìgKidney, 2-5 ìgBrain, 1-2ìgEndothelial cells, 7-12 ìgFibroblasts, 6-8ìgTroubleshooting•Low RNA Yields: Incomplete lysis of samples in RNA Solv Reagent. RNA pellet not completelt dissolved in DEPC-water.pH of diluent used for spectrophotometric analysis is too low.•Degraded RNA: Tissues were not immediately processed or frozen. Inadequate storage of starting material prio to isolation. Inadequate storage of RNA (-5 to -20°C, instead of -60 to -70°C) Trypsin/EDTA was used in dislodging monolayer cells. Buffers or plasticwasre were not RNase-free.Formaldehyde used for denaturing agarose-gel electrophoresis had a pH below 3.0.•Low Abs260/Abs280 ratios: Sample was diluted in water rather than TE. Acidic pH lowers absorbance ratios. Use TE buffer as diluent for readings. Insufficient RNASolv Reagent was used for lysis of sample. Ice incubation in step 2 was not performed. The aqueous phase was contaminated with the phenolic phase.•DNA contamination of RNA: Too little RNASolv Reagent used for sample processing causing inadequate separation of DNA/nucleoprotein complexes from aqueous RNA. The aqueous phase was contaminated with the phenol phase.Technical Support:Omega Bio-tek, USA - call toll-free : 1 888 832 8896References:1. Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156 (1987).2. Chomczynski, P. Biotechniques 15, 532 (1993).For laboratory research use only.CAUTION: Not for diagnostic use. The safety and efficacy ofthis product indiagnostic or other clinical uses has not been established.May,1999 (C). All rights reserved by Omega Bio-tek, Inc.RNA-Solv is a registered mark of Omega Bio-tek, Inc.。

OMEGArna提取试剂盒中文说明

OMEGArna提取试剂盒中文说明E.Z.N.A. T otal RNA Kit I (R6834-01 50 preps)步骤A ．真核细胞和组织自己需要的材料：β－巯基乙醇、70％乙醇（DEPC 水配置）、除RNase 的枪头和EP 管。

材料的最佳量想得到最佳的产量和好的Hibind RNA 柱的纯化效果，选用正确的细胞和组织量是最重要的。

Hibind RNA 柱的最大处理量是可变的，根据细胞和组织的类型。

最大的结合能力是100ug 的RNA 。

TRK 裂解缓冲液的最大裂解能力是1*107个细胞或30mg 的组织。

细胞的步骤1．用500ul 的TRK 裂解缓冲液裂解细胞（≤1*107），使细胞团松散，轻轻弹EP 管或者用枪头吹旋，再用漩涡振荡混匀。

a) 使用前每1ml TRK 裂解缓冲液加入20ul 的β－巯基乙醇。

b) 如果是单层的组织培养细胞（成纤维细胞，内皮细胞等），可以直接在细胞培养器里裂解细胞。

完全吸去培养基后直接加TRK 裂解缓冲液至细胞中。

加700ul 的TRK 到T35细颈瓶或10cm 的培养皿中，更小的培养器加500ul 的TRK 。

将液体布满整个器皿表面，确保细胞裂解完全。

将裂解产物转移至1.5mlEP 管中。

c) 如果细胞是悬液培养，收集细胞（≤1,500rpm or 400*g ）*5min ，弃上清，加入TRK裂解液，漩涡振荡混匀。

2．匀浆化裂解产物“裂解和匀浆化样品”－第四页有详细的说明，如果细胞≤1*105，漩涡振荡1min 。

不完全使样品匀浆化会显著的减少RNA 的产量还容易造成柱子堵塞。

a) 用匀浆器30 s ，使样品匀浆化。

b) 用钝的针头的注射器（0.9mm 直径），至少吹打5次是样品匀浆化。

注射器要除RNase 。

3．将匀浆后的裂解产物转移至Homogenization Spin Column 中，离心，≥1,4000*g, 3min,室温。

将离心收集的流出液转移到新的EP 管中。

细胞或组织样品RNA抽提实验操作SOP

细胞或组织样品RNA抽提实验操作SOP一、样品RNA的抽提:A.进行RNA抽提实验前，RNA操作实验室需紫外灯照射15分钟，关闭紫外灯后开启通风系统抽风15分钟。

B.进入RNA操作实验室后需要佩戴一次性无菌口罩、无菌乳胶手套、实验中尽量避免与同事交谈，防止RNA酶污染；C.实验前将70%乙醇涂布于操作台及器具（移液器等）表面，两分钟后用纸巾擦除。

D.准备实验中放污染材料的器皿、用于RNA操作的耗材（RNAse Free）1ml 枪头、200ul枪头、10ul枪头、1.5ml离心管，用于RNA操作的试剂Trizol Reagent、氯仿（AR）、异丙醇（AR）、无水乙醇（AR）、DEPC H2O，用于进行逆转录实验的试剂MMLV、MMLV 5×Buffer、RNA Inhibitor、Oligo dT、DEPC H2O（注意必须在测定完RNA OD后才可以开始取出配置）；E.一个实验区域在同一时间段只进行一种实验。

F.实验结束后移出个人专用材料（实验记录本）至个人专用区域保管；封好污染材料盛放器皿并移出至污物袋；移出共用器具至专门区域保管；将70%乙醇涂布于操作台或器具表面，两分钟后用纸巾擦除；打开紫外灯照射至少15分钟。

（一）、细胞样品RNA的抽提:G.将贴壁细胞的培养液倒掉，迅速加入1ml trizol，反复吹吸（10次以上）使细胞样品充分裂解。

H.转移到一1.5ml DEPC处理的无菌离心管中，加入500ul氯仿，剧烈震荡混匀，4度12000rpm离心10min。

I.如果上层溶液比较混浊，则转移上清至另一离心管中，重复步骤B。

J.转移上清至另一离心管中（注意不要吸到中间层的蛋白），加入等体积异丙醇，震荡混匀，室温放置2min，4度12000rpm离心10min。

K.轻轻移弃上清，防止吸去沉淀，加入1ml 70%乙醇（注意先加入无水乙醇，然后加入DEPC 水），悬浮沉淀，4度12000rpm离心10min。

RNA提取试剂盒说明书

Binding Capacity
Each HiBind ® RNA column can bind approximately 100 ìg RNA. Using greater than 250 mg fungal tissue in many cases will not drama tical improve yields and sometimes has
E.Z.N.A.™ Fungal RNA Protocol A (Standard Protocol)
Materials to be provided by user ! Microcentrifuge capable of 10,000 x g ! ! ! ! Nuclease-free microfuge tubes 2-mercaptoethanol Absolute (96%-100%) ethanol Isopropyl alcohol (isopropanol) Liquid nitrogen for freezing/disrupting samples Preheat an aliquot (100 ìl per sample) of DEPC-treated water at 65oC.
H iBind
®
R N A C olum ns
H om ogenizer C olum ns 2 m l C ollection Tubes B uffer R FL B uffer SP B uffer R B R N A W ash Buffer II R N A wash Buffer I D E P C W ater Instruction Booklet
Contents
Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Storage and Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Kit Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Before Starting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Fungal RNA Protocol A (Standard Protocol). . . . . . . . . . . . . . . . . . . . 4 Fungal RNA Protocol B (For difficult Samples)). . . . . . . . . . . . . . . . . . 6 On-Membrane DNase Digestion Protocol. . . . . . . . . . . . . . . . . . . . . . 8 RNA Isolation from Arthropods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Quantization of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

罗氏TriPure提RNA

罗氏TriPure法提植物组织RNA一：原理二：试剂准备：异丙醇75%酒精氯仿RNase Water（去离子水）罗氏TriPure三：步骤1：将新鲜或液氮冻存的植物组织样品称量后，迅速转入液氮预冷的研钵中，充分研磨至粉末状，期间不断添加液氮防止RNA降解。

研磨不彻底会影响RNA 回收效率。

每50—100mg加入1ml TriPure。

2：充分混匀（可选步骤：对于含有较多的蛋白质，脂肪等杂物。

4℃12000转离心10min。

取上清加入另一个离心管中。

（此步骤产物可以于-80℃中放置至少一个月，但可能会导致RNA降解或者影响回收效率）3: 所得上清液于15—25℃放置5min。

保证充分混匀4：每1ml TriPure加入0.2ml氯仿。

剧烈震荡15s5:15—25℃放置10min6：4℃ 12000转离心15min.使液体分三成。

无色上清为RNA，红色下层为DNA和蛋白质。

7：转移上清到另一个离心管中。

8：每1ml TriPure加入0.5ml异丙醇，上下颠倒使其充分混匀。

9：15—25℃放置5—10min使RNA沉淀。

4℃ 12000转离心10min。

弃上清10：每1ml TriPure加入1ml 75% 酒精，震荡数秒。

（RNA可在75%酒精4℃中保存一周，-40中保存一年）11：4℃ 7500转离心15min。

弃上清12：加入30ul去离子水重悬。

13：充分混匀RNA。

55—60℃水浴10-15min。

检测1：Nano-Drop2000 超微分光光度计( Thermo 公司)测定OD260 /280值，检测ＲNA 的浓度和纯度2：凝胶电泳（0.8%15min120v）时会产生清晰的28S和18S rRNA条带。

28S：18S=2:1（是否加Marker）三：注意RNA之所以降解，主要有几个因素：1：无处不在的RNase。

做RNA相关的实验，最好有专门的实验室，如果没条件，也要争取弄一片相对独立的空间，开工之前用酒精擦擦台面，枪头，器皿最好是灭菌后新开封的，不要用上次留下的。

翻译好的罗氏公司Tunel试剂盒操作说明书

罗氏 (Roche)公司 Tunel 试剂盒操作说明书(In situ cell death detection kit-POD法)一、原理：TUNEL （TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA 的断裂情况。

其原理是荧光素（ fluorescein）标记的 dUTP 在脱氧核糖核苷酸末端转移酶（ TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂 DNA 的3’-OH 末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与 HRP 底物二氨基联苯胺（DAB ）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有 DNA 断裂，因而没有 3‘-OH 形成，很少能够被染色。

本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。

还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。

二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1 号（蓝盖） Enzyme Solution 酶溶液： TdT 10×、2号（紫盖） Label Solution 标记液：荧光素标记的 dUTP 1×、3号（棕瓶） Converter-POD:标记荧光素抗体的 HRP；自备试剂： PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB 工作液（临用前配制， 5 μl 20 ×DAB+1 μL 30%H2O2+94 μl PBS）、Proteinase K工作液（ 10-20 μg/ml in 10 mM Tris/HCl ，pH 7.4-8）或细胞通透液（0.1% Triton X-100 溶于 0.1% 柠檬酸钠，临用前配制）、苏木素或甲基绿、 DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl ，pH 7.5， 10 mM MgCl2 ，1 mg/ml BSA ）等。

RNA抽提详细步骤

RNA抽提指南(TRIZOL法)RNA抽提指南(TRIZOL法)注意事项：* 全程佩戴一次性手套。

皮肤经常带有细菌和霉菌，可能污染RNA的抽提并成为RNA酶的来源。

培养良好的微生物实验操作习惯预防微生物污染。

* 使用灭菌的，一次性的塑料器皿和自动吸管抽提RNA，避免使用公共仪器所导致的RNA酶交叉污染。

例如，使用RNA探针的实验室可能用RNA酶A 或T1来降低滤纸上的背景，因而某些非一次性的物品（如自动吸管）可能富含RNA酶。

* 在TRIZOL 中，RNA 是隔离在RNA 酶污染之外的。

而对样品的后续操作会要求用无RNA 酶的非一次性的玻璃器皿或塑料器皿。

玻璃器皿可以在150°C 的烘箱中烘烤4 小时。

塑料器皿可以在0.5 M NaOH 中浸泡10 分钟，用水彻底漂洗干净后高压灭菌备用。

抽提步骤：1．匀浆化作用通过离心来沉淀细胞后，弃上清，用移液管加TRIZOL试剂反复吹打来裂解细胞至均一通亮的液态后，将匀浆样品在15—30°C 条件下孵育5 分钟以使核蛋白体完全分解。

（每5—10×106的动物细胞，植物或酵母菌细胞或每1×107 细菌加1ml的TRIZOL。

在加入TRIZOL前应避免洗涤细胞因为那样会增加mRNA降解的可能性。

）2．分离阶段每1 mlTRIZOL 加0.2 ml 氯仿。

盖紧样品管盖，用手用力摇晃试管15 秒并将其在30°C 下孵育2—3 分钟。

在2—8°C 下以不超过12,000×g 的离心力高速冷冻离心15 分钟。

离心后混合物分成三层：下层红色的苯酚-氯仿层，中间层，上层无色的水样层。

RNA 无一例外地存在于水样层当中。

水样层的容量大约为所加TRIZOL 容量的60%3．RNA的沉淀将水样层转移到一干净的试管中，通过将水样层和异丙醇混合来沉淀RNA。

最初均化时的每1 m lTRIZOL 对应0.5 ml 异丙醇。

Omega公司RNA提取试剂盒说明书

RNA-Solv Reagent®RNA Isolation SolventWARNING: This reagent is toxic if swallowed and in contact with skin. Causes burns. After contact with skin, wash immediately with copious amounts of mild detergent and water. If you feel sick, seek medical advice at once and Quote UN2821.Product No:R6830-00 (5 ml)R6830-01 (100 ml)R6830-02 (200 ml)Storage Conditions:RNA-Solv is stable for at least 24 months®when stored at 2°C-8°C and yields reproducible results.IntroductionRNA-Solv® Reagent is a reagent system for the isolation of total RNA from cells and tissues. The reagent, a single-phase solution consisting of phenol and guanidine isothiocyanate, is modification of the single-step RNA isolation method developed by Chomczynski and Sacchi (1).The sample is homogenized and lysed in RNA-Solv® Reagent which maintains the integrity of the RNA, while disrupting and denaturing endogenous RNases and other cellular components. Extraction of the lysate with chloroform further denatures proteins and separates the mixture into an organic and an aqueous phase. RNA remains exclusively in the aqueous phase, and is subsequently recovered by isopropanol.This method is suitable for small quantities of tissue (<100 mg) and cells (<5 X10), and large quantities of tissue ( up to1 g) and cells 6(<10 ), of human, animal, plant, or bacterial origin. The simplicity 8of the RNA-Solv® Reagent method allows simultaneous processing of a large number of samples. The entire procedure can be completed in one hour. Total RNA prepared in this manner can be used for Northern blot analysis, dot blot hybridization, poly(A) + selection, in vitro translation, RNase protection assay, and molecular cloning. For use in amplification by thermal cycling, treatment of the isolated RNA with RNase-free DNase I is recommended when the two amplimers lie within a single exon.Supplied By User•Chloroform (no isoamyl alcohol added)•Isopropyl alcohol•80% Ethanol (in DEPC-treated water)•RNase-free water•Tabletop centrifuge capable of 12,000 x g at room temperatureGeneral Notes Regarding RNase ContaminationWhenever working with RNA :•Always wear disposable gloves and change gloves frequently.•Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases.•In the presence of RNA-Solv® Reagent, RNA is protected from RNase contamination. Downstream sample handling requires that nondisposable glassware or plasticware be RNase-free.•Use only DECP-treated buffers. Add DEPC to a final concentration of 0.1%, incubate at 37C for 2 hours, andoautoclave at 121C. Do not add DEPC to Tris buffers. Suchobuffers must be prepared by using DECP-water.PrecautionUse only disposable polypropylene tubes for small samples and glass Corex tubes for larger samples. All tubes must be able to withstand 12,000 x g . Polystyrene tubes may crack with chloroform Before StartingA.Small Samples :To isolate RNA from very small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) of samples in 0.8 mL of RNA-Solv®, and add 1 mg RNase-free glycogen or yeast tRNA as carrier. This will improve yields obtained with precipitation.B.Difficult Animal Samples: Specimens containing large amounts of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and sperm, will require the following modification. After lysis/homogenization in RNA-Solv® Reagent, centrifuge at 12,000 x g for 10 minutes at room temperature to remove insoluble debris. Often a precipitate forms at the bottom of the tube, but with fatty tissue, a lipid layer will also form above the aqueous phase. The supernatant will contain the RNA and must be carefully transferred to a fresh 1.5 ml microfuge tube before proceeding.C. Interruption the procedure:Following lysis in RNA-Solv®Reagent and before addition of chloroform, samples can be stored at -70C for up to 3 months. In addition, once the RNA is oprecipitated in isopropanol, the pellet may be stored at -20C or -o70C for up to 1 year.oRNA-Solv Protocol for Total RNA Isolation®CAUTION: When working with RNA-Solv® Reagent use gloves and eye protection (safety goggles) and avoid contact with skin or clothing. Work in a chemical fume hood to avoid inhaling vapor. Unless otherwise noted, all steps are to be carried out at room temperature (20C-25C).o o1. Homogenization and lysis of samples: follow either method belowa) Tissue SamplesHomogenize tissue samples in 1 mL of RNA-Solv® Reagent per 50-100 mg of tissue using an appropriate mechanical homogenizer. Alternatively one can pulverize tissue in liquid nitrogen with mortar and pestle and transfer the powder to a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (Eppendorf, Cat No. 0030 120.973; VWR, Cat No. KT 749520-0000). The sample volume should not exceed 10% of the volume of RNA-Solv® Reagent used.b) Cells Grown in SuspensionPellet cells by centrifugation. Lyse cells in RNA-Solv® Reagent by repetitive pipetting. Use 1 mL of the reagent per 5-10 x 10of6 animal, plant or yeast cells, or per 1 x 10 bacterial cells. Washing8cells before addition of RNA-Solv® Reagent should be avoided as this increases the possibility of mRNA degradation and RNase contamination. For plant, fungal, and yeast cells mechanical or enzymatic homogenization may be required. Also, for plant, fungal, and yeast cells, we recommend the use of the E.Z.N.A.® Plant (R6627),Fungal (R6640), and Yeast (R6670) RNA Kits from Omega Bio-tek.c) Cells Grown in MonolayerLyse cells directly in a culture dish by adding 1 mL of RNA-Solv®Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a blue pipette tip. The amount of RNA-Solv®Reagent added is based on the area of the culture dish (~1 mL per 10 cm ). An insufficient amount of RNA-Solv® Reagent may result2in contamination of the isolated RNA with DNA. Always use more RNA-Solv® Reagent if in the lysate is too viscous to aspirate with a pipette.2. Add 0.2 mL of chloroform per 1 mL of RNA-Solv® Reagent. Cap sample tubes securely and vortex vigorously for 15 seconds. Incubate on ice for 10 minutes. This step is critical - do not change it.3. Centrifuge the samples at no more than 12,000 x g for 15 minutes 4E C.The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA remains entirely in the aqueous phase.4. Precipitation of RNA.Transfer no more than 80% of the aqueous phase to a fresh tube, and discard the lower organic phase. Precipitate the RNA from the aqueous phase by adding 500ìl of isopropyl alcohol per 1 mL of RNA-Solv® Reagent used for the initial homogenization. Incubate samples at room temperature 10 minutes and centrifuge at no more than 12,000 x g for 10 minutes also at room temperature.Carbohydrate-rich samples: Plant samples of high polysaccharide content or animal tissues rich in glycosaminoglycans (proteoglycans) require the following modified precipitation method for obtaining pure RNA. Prepare Buffer A ( 1.2 M sodium chloride, 800 mM sodium citrate). Following step 3, add to the aqueous phase 0.3 ml isopropanol followed by 0.3 ml Buffer A per 1 ml RNA Solv ® Reagent used in step 1. Vortex to mix and centrifuge at no more than 12,000 x g for 10 minutes at room temperature. This high salt precipitation will reduce co-purification of complex carbohydrates.5. Wash RNA pellet. Discard the supernatant and wash the RNA pellet once with 1 ml 80% ethanol. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at room temperature.6.Reconstitute RNA. Carefully aspirate and discard the ethanol and briefly AIR DRY the RNA pellet for 2-5 minutes at room temperature. Do not use centrifugal devices equipped with a vacuum source as over-drying will lead to difficulty in re-dissolving RNA in water. Dissolve RNA in RNase-free water - a 5 minute incubation at 60 °C may be required. RNA can also be reconstituted in 100% formamide (deionized) and stored at -70°C.RNA is now suitable for RNase protection, northern analysis and reverse transcriptase reactions. For isolation of poly(A)+ RNA an additional ethanol precipitation is required. Add 1/8 X volume of RNase-free 3M NaAc, pH 6.0 followed by 2.5 X volume absolute ethanol. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge at 12,000 x g for 10 min at room temperature and discard the supernatant. Wash the pellet as before and reconstitute in DECP-treated water.Determination of Yield and QualityUV spectrophotometric analysis of the purified RNA is required for obtaining yield. To do so, dilute the RNA in an appropriate volume of TE buffer, pH 8.0 (not water; RNA yields low Abs ratio values if dissolved in acidic buffers) and measure absorbance at 260 nm and at 280 nm. RNA Conc = 40 ìg/ml X Dilution factor X Abs 260 nmTypical Abs 260 nm/ 280 nm ratios of 1.7-1.9 are obtained with the protocol. Yields vary depending of type and amount of starting material, and on condition of storage prior to processing. For assessing the quality of RNA, we recommend you perform denaturing agarose gel electrophoresis to confirm the integrity of purified material. Invariably, the full spectrum of RNAs, including 4S and 5S species are purified with RNA-Solv® Reagent.Expected Yields per 1 mg tissue or 10 cells:6Liver and spleen, 5-10 ìgKidney, 2-5 ìgBrain, 1-2ìgEndothelial cells, 7-12 ìgFibroblasts, 6-8ìgTroubleshooting•Low RNA Yields: Incomplete lysis of samples in RNA Solv Reagent. RNA pellet not completelt dissolved in DEPC-water.pH of diluent used for spectrophotometric analysis is too low.•Degraded RNA: Tissues were not immediately processed or frozen. Inadequate storage of starting material prio to isolation. Inadequate storage of RNA (-5 to -20°C, instead of -60 to -70°C) Trypsin/EDTA was used in dislodging monolayer cells. Buffers or plasticwasre were not RNase-free.Formaldehyde used for denaturing agarose-gel electrophoresis had a pH below 3.0.•Low Abs260/Abs280 ratios: Sample was diluted in water rather than TE. Acidic pH lowers absorbance ratios. Use TE buffer as diluent for readings. Insufficient RNASolv Reagent was used for lysis of sample. Ice incubation in step 2 was not performed. The aqueous phase was contaminated with the phenolic phase.•DNA contamination of RNA: Too little RNASolv Reagent used for sample processing causing inadequate separation of DNA/nucleoprotein complexes from aqueous RNA. The aqueous phase was contaminated with the phenol phase.Technical Support:Omega Bio-tek, USA - call toll-free : 1 888 832 8896References:1. Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156 (1987).2. Chomczynski, P. Biotechniques 15, 532 (1993).For laboratory research use only.CAUTION: Not for diagnostic use. The safety and efficacy ofthis product indiagnostic or other clinical uses has not been established.May,1999 (C). All rights reserved by Omega Bio-tek, Inc.RNA-Solv is a registered mark of Omega Bio-tek, Inc.。

ROche 提RNA的说明书

For general laboratory use. Not for use indiagnostic procedures. FOR IN VITRO USE ONLY.High Pure RNA Tissue KitVersion February 2008For isolation of total RNA from tissueCat. No. 12 033 674 001Kit for 50 isolations Store the kit at ؉15 to ؉25°CTable of Contents1.What this Product Does (3)Number of Isolations 3 Kit Contents 3 Storage and Stability 3 Additional Equipment and Reagents Required 4 Application 4 Assay Time 4 2.How To Use this Product (5)2.1Before You Begin 5Precautions 5 Sample Material 5 Handling and Storage of Starting Material 5 Disruption and Homogenization 5Disruption and Homogenization using Rotor-Stator Homogenizers 6 Disruption using a Mortar and Pestle 6 Homogenization using a Syringe and Needle 6 Preparation of Working Solutions 6 2.2Experimental overview 72.3Protocol for the Isolation of RNA from Tissue 83.Results (10)Purity 10 Expected Yield 104.Troubleshooting (11)5.Additional Information on this Product (13)How this Product Works 13 Basic Steps 13 References 13 Quality Control 14 6.Supplementary Information (15)6.1Conventions 15Text Conventions 15 Symbols 15 6.2Changes to previous version 15 6.3Ordering Information 16 6.4Disclaimer of License 176.5Trademarks 177.Quick Reference Protocol.................................................................................................. 18P R O T O C O LPROTOCOL21.What this Product DoesNumber ofIsolationsThe kit is designed for 50 isolations of total RNA from tissue.Kit Contents N All solutions are clear, and should not be used when precipitates haveformed. Warm the solutions at +15 to +25°C or in a 37°C waterbath untilthe precipitates have dissolved.Storage and Stability The High Pure RNA Tissue Kit components must be stored at +15 to +25°C. If properly stored all kit components are stable until the expiration date printed on the label.N Please note, that improper storage at +2 to +8°C (refrigerator) or -15 to -25°C (freezer) will adversely impact nucleic acid purification when precipi-tates form in the solutions. Therefore, High Pure isolation kits are always shipped at +15 to +25°C.After dissolving DNase I lyophilizate in Elution Buffer, store the solution ali-quoted at –15 to –25°C; this solution is stable for 12 months.Vial/Cap Label Contents / Function1greenLysis/BindingBuffer•25ml•[4,5 M guanidine-HCl, 100 mM sodium phos-phate pH 6.6 (25°C)].2whiteDNase I•recombinant•Lyophilizate•10 kU DNase I•For digesting residual contaminating DNA3whiteDNase Incuba-tion Buffer•10ml•1 M NaCl, 20 mM Tris-HCl, 10 mM MnCl2,pH 7.0 (25°C)4blackWash Buffer I•33 ml, add 20 ml absolute ethanol•5 M guanidine-HCl, 20 mM Tris-HCl, pH 6.6(25°C) final concentration after addition ofethanol5blueWash Buffer II•10 ml add 40 ml absolute ethanol•20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (25°C)final concentrations after addition of ethanol 6colorlessElution Buffer•30 ml•Nuclease-free, sterile, double distilled water 7High Pure FilterTubesOne bag with 50 polypropylene tubes withtwo layers of glass fiber fleece, for use of up to700 µl sample volume.8Collection Tubes One bag with 50 polypropylene tubes (2 ml).Additional Equipment and Reagents Required •Absolute ethanol•Standard tabletop microcentrifuge capable of 13,000 × g centrifugal force (e.g., Eppendorf 5415C or equivalent)•Microcentrifuge tubes, 1.5 ml, sterile•Mortar and Pestle or Rotor-Stator Homogenizer (e.g., Ultra Turrax)Application The High Pure RNA Tissue Kit is designed to purify total, intact total RNA from tissue samples (e.g., mouse liver, spleen, lung, heart) for use in research. Theisolated RNA may be used directly as template for RT-PCR, northern blotting,and RNAse protecion assays.Assay Time Total time: approx. 30 min with rotor-stator homogenization. Add additional time when using alternative disruption methods.1.What this Product Does, continued42.How To Use this Product2.1Before You BeginPrecautions N Lysis/Binding Buffer and Wash Buffer I contain guanidine hydrochloridewhich is an irritant. Always wear gloves and follow standard safety pre-cautions to minimize contact when handling.•Do not let these buffers touch your skin, eyes, or mucous membranes. Ifcontact does occur, wash the affected area immediately with large amountsof water; otherwise, the reagent may cause burns. If you spill the reagent,dilute the spill with water before wiping it up.•Never store or use the Binding Buffer near human or animal food.•Always wear gloves and follow standard safety precautions when handlingthese buffers.Sample Material Solid tissue (e.g., mouse liver, spleen, lung, heart), 1 – 10 mg (for mortar/pestle disruption) or 1 – 25 mg (for rotor-stator homogenization).Handling and Storage of Starting Material To avoid any degradation of RNA in sample material by intracellular RNases follow these guidelines:•Process the sample material as soon as collected•If storage of sample material is required, flash freeze in liquid nitrogen and store at -60°CN Do not allow frozen tissue material to thaw during handling (e.g., weighing). The relevant procedures should be carried out as quickly as possible.•Samples can also be stored at -60° C or below in Lysis/Binding Buffer after disruption and homogenization.L Yields may vary depending on storage time.N Use sterile disposable polypropylene tubes and tips in order to avoid RNase contamination. Wear gloves during the assay.Disruption and Homogenization •Efficient disruption and homogenization of the starting material is essential for intra-cellular RNA isolation procedures from tissues.•The complete disruption of cell walls and plasma membranes of cells and organelles is absolutely required to release all the RNA contained in the sam-ple. Incomplete disruption results in significantly reduced yields. •Homogenization is necessary to reduce the viscosity of the cell lysates pro-duced by disruption. Homogenization shears the high molecular weight genomic DNA and other high molecular weight cellular components to cre-ate a homogeneous lysate.•Incomplete homogenization results in significantly reduced yields. Some dis-ruption methods simultaneously homogenize the sample, while others require an additional homogenization step.Disruption and Homogenization using Rotor-Stator Homogenizers In the presence of lysis buffer, rotor-stator homogenizers thoroughly disrupt and simultaneously homogenize tissues in 5 – 90 s, depending on the viscosity of the sample. By a combination of turbulence and mechanical shearing, the sample will be disrupted and homogenized.Keep foaming of the sample to a minimum by keeping the tip of the homoge-nizer submerged and holding the immersed tip to one side of the tube.Disruption using a Mortar and Pestle N This step is for disruption, only. Homogenization must be performed sepa-rately!For disruption using a mortar and pestle, freeze the sample immediately in liq-uid nitrogen and grind to a fine powder under liquid nitrogen. Transfer the fro-zen tissue powder into a tube with 350 ␮l Lysis/Binding Buffer. Continue as quickly as possible with the homogenization step.Homogenization using a Syringe and Needle After disruption, the tissue lysate can be homogenized using a syringe and needle. High molecular weight DNA is sheared by passing the lysate through a 20-gauge needle attached to a sterile plastic syringe. Pass the lysate through the needle, at least 5–10 times, or until a homogenous lysate is achieved.Preparation of Working Solutions Beside the ready-to-use solutions supplied with this kit, you will need to pre-pare the following working solution:ContentReconstitution/PreparationStorage andStabilityFor use inDNase I(Vial 2; whitecap)Dissolve DNase I in550 ␮l ElutionBuffer and mixthouroghly.Store aliquots at -15to -25°C, stable for 12month.Step 6:Purifies the RNAfrom residualgenomic DNAWash Buffer I(Vial 4; blackcap)Add 20 ml absoluteethanol to WashBuffer.N Mark and datebottle onceethanol hasbeen added.Store at +15 to+25°C.Stable until expirationdate printed on kitlabel.Step 7:Removes Inhibi-torsWash BufferII (Vial 5; bluecap)Add 40 ml absoluteethanol to WashBuffer.N Label and datebottle accord-ingly after add-ing ethanol.Store at +15 to+25°C.Stable until expirationdate printed on kitlabel.Step 8 and 9:Purifies the tem-plate DNA fromresidual impuri-ties2.1Before You Begin, continued62.2Experimental overviewLysate supernatantAdd 0.5 volumes ethanol abs.(tissue homogenized in lysisbuffer)Mix and apply lysate (max. 700 ␮l at atime) to a High Pure filter tube, centri-fuge at 13 000 × g for 30 s (repeat ifthe lysate volume is more than 700 ␮l)Add 90 ␮l DNase IncubationBuffer/10 ␮l DNase working Discard flowthroughsolutionIncubate 15 min at +15 to +25° CAdd 500 ␮l Wash Buffer ICentrifuge at 8,000 × g for15sDiscard flowthrough Add 500 ␮l Wash Buffer IICentrifuge at 8,000 × g for15sDiscard flowthrough Add 300 ␮l Wash Buffer IICentrifuge at 13,000 × g for 2minDiscard flowthrough Add 100 ␮l Elution BufferCentrifuge at 8,000 × g for1minPure total RNA82.3Protocol for the Isolation of RNA from Tissue³Depending on the disruption and homogenization method, add one of the following to a nuclease-free 1.5 ml microcentrifuge tube:•add 400 ␮l Lysis/Binding Buffer and the appropriate amount of fro-zen tissue (max. 20 – 25 mg); disrupt and homogenize the tissue using a rotor-stator homogenizer (e.g., Ultra Turrax).L Increasing the volume of Lysis buffer may be required to facilitatehandling and minimize loss.alternatively:•add 400 ␮l Lysis/Binding Buffer and the appropriate amount of tis-sue-powder (grinded with a mortar and pestle) and pass this lysate 5 – 10 times through a 20-gauge needle fitted to a syringe.L For optimal yield do not exceed 10 mg tissue.·Centrifuge lysate for 2 min at maximum speed in a microcentrifuge and use only the collected supernatant for subsequent steps.L Sometimes very small amounts of insoluble material will be presentmaking this pellet invisible.»Add 200 ␮l (0.5 volumes) absolute ethanol to the lysate supernatant and mix well.L If some lysate is lost during homogenization, reduce volume of eth-anol accordingly.¿Combine the High Pure Filter Tube with the Collection Tube and pipet the entire sample in the upper reservoir (maximal volume 700 ␮l).´•Centrifuge 30 s at maximal speed (13, 000 × g ) in a standard table top microcentrifuge.N After this centrifugation step, the glass fleece must be dry; if it lookswet, the centrifugation time must be increased.²•Remove the Filter Tube from the Collection Tube, discard the flowthrough liquid.•Combine the Filter Tube with the used Collection Tube.¶•For each isolation pipet 90 ␮l DNase Incubation Buffer (white cap)into a sterile 1.5 ml reaction tube, add 10 ␮l DNase I working solution, mix.•Pipet the solution to the upper reservoir of the Filter Tube.•Incubate for 15 min at +15 to +25°C.º•Add 500 ␮l Wash Buffer I (black cap) to the upper reservoir of the Fil-ter Tube.•Centrifuge 15 s at 8,000 × g .•Remove the Filter Tube from the Collection Tube, discard the flowthrough liquid.•Combine the Filter Tube with the used Collection Tube.¾•Add 500 ␮l Wash Buffer II (blue cap) to the upper reservoir of the Filter Tube.•Centrifuge 15 s at 8,000 × g and discard the flowthrough.•Combine the Filter Tube with the used Collection Tube.P r o t o c o l f o r t h e I s o l a t i o n o f R N A f r o m T i s s u eµ•Add 300 ␮l Wash Buffer II (blue cap) to the upper reservoir of the Filter Tube.•Centrifuge 2 min at full speed (approx 13, 000 × g ).L The extra centrifugation time ensures removal of residual WashBuffer.¸•Carefully remove the column from the collection tube so that the col-umn does not contact the flowthrough as this will result in carryover of ethanol.N Residual ethanol may interfere with subsequent reactions.•Insert the Filter Tube into a nuclease free, sterile 1.5 ml microcentri-fuge tube.¹•Add 100 ␮l Elution Buffer to the upper reservoir of the Filter Tube.•Centrifuge the tube assembly for 1 min at 8,000 × g .ƸThe microcentrifuge tube now contains the eluted RNA.L Either use 10 ␮l of the eluted RNA directly in RT-PCR or store theeluted RNA at -80°C for later analysis.2.3Protocol for the Isolation of RNA from Tissue, continuedProtocol for the isolation of RNA from Tissue3.ResultsPurity Purified RNA is free of DNA, nucleases and all cellular and sample compo-nents that interfere with RT-PCR.The absence of contaminating DNA is examined by a PCR without a preced-ing RT-reaction; no amplification product is obtained.The integrity and size distribution of total RNA purified with the High PureRNA Tissue Kit has been checked by denaturing agarose gel electrophoresisand ethidiumbromide staining. The relevant ribosomal species appear assharp bands on the stained gel. 28 S ribosomal RNA band should be presentat approximately twice the amount of the 18 S rRNA (mouse-tissue).Expected Yield The concentration and purity of RNA can be determined by measuring the absorbance at 260 nm and 280 nm in a spectrophotometer. An absorbance of1 unit at 260 nm corresponds to 40 ␮g of RNA per ml. The ratio between theabsorbance values at 260 and 280 nm gives an estimate of RNA purity andshould be 2.0 (RNA diluted in 20 mM Tris-HCl, pH 7.5).The yield of total RNA depends on the starting material and varies dependingon the amount of tissue used and the kind of disruption method applied. Theyield with mouse muscle tissue could not be determined by spectrophotome-ter, but isolated RNA resulted in a specific RT-PCR signal.type of mouse tissue yield [␮g/mg]–2.8liver 0.51.0–kidney 0.53.0spleen 0.5––0.5lung 0.3heart0.3muscle n.d.104.TroubleshootingPossible Cause RecommendationLow nucleic acid yield or purity Kit stored under non-opti-mal conditions.Store kit at +15 to +25°C at all times uponarrival.Buffers or other reagentswere exposed to conditionsthat reduced their effective-ness.•Store all buffers at +15 to +25°C.•Close all reagent bottles tightly after each useto preserve pH, stability, and freedom fromcontamination.•After any lyophilized reagent is constituted,aliquot it and store the aliquot at -15 to -25°C. Ethanol not added to WashBuffer and InhibitoryRemoval Buffer•Add absolute ethanol to the buffers beforeusing.•After adding ethanol, mix the buffers well andstore at +15 to +25°C.•Always mark Wash Buffer vial and InhibitoryRemoval Buffer vial to indicate whether etha-nol has been added or not.Reagents and samples notcompletely mixed.Always mix the sample tube well after addi-tion of each reagent.Tissue stored and handled inless than optimal conditionsUse fresh tissue and disrupt immediately orflash frozen tissue stored at -60°C or below.Frozen tissue should not be allowed to thawduring handling prior to disruption in Lysis/Binding Buffer.Ethanol not added to thelysate in step 3Addition of 0.5 volume of absolute ethanol tothe lysate is necessary to promote selectivebinding of RNA to the glass fibers.High levels of RNase activity •Be careful to create an RNase-free workingenvironment.•Process starting material immediately or storeit at -80°C until it can be processed.•Use eluted RNA directly in downstream pro-cedures or store it immediately at -80°C.Tissue homoge-nate is viscous and difficult to pipet, low RNA yield Insufficient disruption orhomogenizationAdd 350 ␮l of Lysis/Binding Buffer and repeathomogenization step to reduce viscosity.Too much starting material Reduce amount of starting material and/orincrease the amount of Lysis/Binding Buffer.Clogged filter tube Insufficient disruption and/or homogenizationE.g., increase the disruption time for therotorstator homogenizer or pass throughsyringe/needle additional times.Too much starting material Reduce amount of starting material and/orincrease the amount of Lysis/Binding Buffer. 12DNA contamina-tionLysis/Binding Buffer not completely removed from the glass fleeceIncrease centrifugation time in step 5.Absorbance (A 260 nm ) reading of product too high Glass fibers, which might coelute with nucleic acid, scatter light1. Remove High Pure Filter Tube from tube containing eluted sample and spin sample for 1 min at maximum speed.2. Transfer supernatant into a new tube without disturbing the glass fibers at the bottom of the original tube.Samples “pop” out of wells in agarose gelsEluate contains ethanol (from the Wash Buffer)1. After the wash step, do not let theflowthrough touch the bottom of the High Pure Filter Tube.2. Empty collection tube, reinsert filter tube in emptied collection tube, and recentrifuge for 30 s.Possible CauseRecommendation4.Troubleshooting, continued5.Additional Information on this ProductHow this Product WorksIsolating intact RNA is a prerequisite for the analysis of gene expression. Fre-quently applied techniques like reverse transcriptase-PCR (RT-PCR), northern blotting, and RNase protection require the use of intact undegraded RNA. Tis-sue samples are disrupted and homogenized in the presence of a strong denaturing buffer containing guanidine hydrochloride to instantaneously inac-tivate RNases, and to ensure isolation of intact RNA. After adding ethanol RNA binds selectively to a glass fiber fleece in the presence of a chaotropic salt (guanidine HCl). Residual contaminating DNA is digested by DNase I, applied directly on the glass fiber fleece. During a series of rapid “wash-and-spin”steps to remove contaminating cellular components the RNA remains bound to the glass fiber fleece. Finally, low salt elution removes the nucleic acids from the glass fiber. The process does not require RNA precipitation, organic sol-vent extractions, or extensive handling of the RNA.Basic StepsReferences1Vogelstein B et al. (1979) Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA 76, 615-619.2Evtimova S et al. (2003) Identification of Genes Associated with the Invasive Status of Human Mammary Carcinoma Cell Lines by Transcriptional Profiling Tumor. Biology 24,189–198.3Kleiman SE et al. (2003) Members of the CDYfamily have different expression patterns:CDY1transcripts have the best correlation with complete spermatogen-esis. Hum. Genet. 113, 486-492.4Lewin E et al. (2003) Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats. Kidney International 64, 63-70.5Dalm VASH et al. (2004) Distribution pattern of somatostatin and cortistatin mRNA in human central and peripheral tissues. Clinical Endocrinology 60, 625-629.6Gertler R et al. (2004) Telomere Length and Human Telomerase Reverse Tran-scriptase Expression As Markers for Progression and Prognosis of Colorectal Carcinoma. J. Clinical Oncology 22, 1807-1814.ቢSamples (max. 25 mg tissue) are disrupted in Lysis/Binding Buffer and homogenized.ባRNA is isolated by binding to the glass fibers pre-packed in the High Pure Filter Tube.ቤResidual contaminating DNA is digested with DNase I.ብBound RNA is washed, thereby purified from salts, proteins and other cellular impurities.ቦElution of the purified RNA with Elution Buffer.5.Additional Information on this Product, continuedQuality Control•Tissue is disrupted and homogenized in Lysis/Binding Buffer and purified as described.•RNA yield is determined by measuring the optical density at 260 nm.•Integrity and size distribution are examined by the banding pattern of riboso-mal RNA in a denaturing agarose gel.•10 ␮l of the RNA eluate is used in first strand synthesis with reverse tran-scriptase M-MuLV and p(dT)15 as primer. In the following PCR, accom-plished with Expand* High Fidelity PCR System and specific primers forglycerinaldehyde 3-phosphate dehydrogenase (GAPDH), the expectedamplification product of 983 bp is obtained.•Absence of contaminating DNA is examined by a PCR without preceding RT-reaction; no amplification product is obtained.* available from Roche Applied Science146.Supplementary Information6.1ConventionsText Conventions To make information consistent and memorable, the following text conven-tions are used in this Instruction Manual:SymbolsIn this Instruction Manual, the following symbols are used to highlight impor-tant information:6.2Changes to previous versionNew Disclaimer of LicenseText Convention UsageNumbered stages labeled ቢ, ባ, etc.Stages in a process that usually occur in the order listedNumbered instructions labeled ³, ·, etc.Steps in a procedure that must be performed in the order listedAsterisk *Denotes a product available from Roche Applied Science.SymbolDescriptionL Information Note:Additional information about the current topic or procedure.NImportant Note:Information critical to the success of the procedure or use of the product. 166.3Ordering InformationRoche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals,please visit and bookmark our home page, ,and our Special Interest Sites including:•Nucleic Acid Isolation and Purification:/napure •PCR - Innovative Tools for Amplification:/pcr ProductPack SizeCat. No.Associated Kits High Pure RNA Isolation Kit50 purifications 11 828 665 001High Pure PCR Product Purification Kit 50 purifications 250 purifications 11 732 668 00111 732 676 001High Pure PCR T emplate Preparation Kit 100 purifications 11 796 828 001First Strand cDNA Synthesis Kit for RT PCR (AMV)30 reactions 11 483 188 001LightCycler ®Kits for RT-PCRLightCycler ® RNA Master HybProbe 1 kit (96 reactions)03 018 954LightCycler ® RNA Master SYBR Green I 1 kit (96 reactions)03 064 760 001LightCycler ®RNA Amplification Kit HybProbe 1 kit (96 reactions)12 015 145 001LightCycler ® RNA Amplification Kit SYBR Green I1 kit (96 reactions)12 015 137 001LightCycler ® Control Kit RNA 1 kit (50 reactions)12 158 841 001High Pure PCR Product Purification Kit50 purifications 250 purifications 11 732 668 00111 732 676 001Single reagents Reverse Transcriptase, M-MuL V 500 U 11 062 603 001Reverse Transcriptase AMV 500 U 1,000 U 11 495 062 00110 109 118 001Protector RNase Inhibitor 2000 U 10 000 U 03 335 399 00103 335 402 001T ranscriptor Reverse Transcriptase250 U 500 U 2000 U03 531 317 00103 531 295 00103 531 287 001T ranscriptor First Strand cDNA Synthesis Kit1 kit (50 reactions)04 379 012 001Primer for cDNA Synthesis p(dT)15 1 A 260 nm unit (40 g, 8 mmol)10 814 270 001Expand Reverse Transcriptase (not available in USA)1,000 U 5,000 U11 785 826 00111 785 834 001Expand High Fidelity PCR System100 U 500 U (2 × 250 U)2,500 U (10 × 250 U)11 732 641 00111 732 650 00111 759 078 001Titan One Tube RT-PCR System25 reactions 100 reactions11 888 382 00111 855 476 0016.4Disclaimer of LicenseNOTICE TO PURCHASER:This is a product licensed under patents owned by Qiagen.6.5TrademarksHIGH PURE, EXPAND and TITAN are Trademarks of Roche.Ultra Turrax is a Trademark of IKA Analysentechnik GmbH, Germany.Triton is a Trademark of Rohm & Haas Company, Philadelphia, PA. USA.SYBR is a Trademark of Molecular Probes Inc., Eugene, OR, USA.Other brands or product names are trademarks of their respective holders.7.Quick Reference Protocol³Depending on the disruption and homogenization method, add one ofthe following to a nuclease-free 1.5 ml microcentrifuge tube:•add 400 ␮l Lysis/Binding Buffer and the appropriate amount of fro-zen tissue (max. 20 – 25 mg); disrupt and homogenize the tissueusing a rotor-stator homogenizer (e.g., Ultra Turrax).L Increasing the volume of Lysis buffer may be required to facilitatehandling and minimize loss.alternatively:•add 400 ␮l Lysis/Binding Buffer and the appropriate amount of tis-sue-powder (grinded with a mortar and pestle) and pass this lysate5 – 10 times through a 20-gauge needle fitted to a syringe.L For optimal yield do not exceed 10 mg tissue.·Centrifuge lysate for 2 min at maximum speed in a microcentrifugeand use only the collected supernatant for subsequent stepsL Sometimes very small amounts of insoluble material will be presentmaking this pellet invisible.»Add 200 ␮l (0.5 volumes) absolute ethanol to the lysate supernatantand mix well.L If some lysate is lost during homogenization, reduce volume of eth-anol accordingly.¿Combine the High Pure Filter Tube with the Collection Tube and pipetthe entire sample in the upper reservoir (maximal volume 700 ␮l).´•Centrifuge 30 s at maximal speed (13, 000 × g) in a standard table topmicrocentrifuge.N After this centrifugation step, the glass fleece must be dry; if it lookswet, the centrifugation time must be increased.²•Remove the Filter Tube from the Collection Tube, discard theflowthrough liquid.•Combine the Filter Tube with the used Collection Tube.¶•For each isolation pipet 90 ␮l DNase Incubation Buffer (white cap)intoa sterile 1.5 ml reaction tube, add 10 ␮l DNase I working solution, mix.•Pipet the solution to the upper reservoir of the Filter Tube.•Incubate for 15 min at +15 to +25°C.º•Add 500 ␮l Wash Buffer I (black cap) to the upper reservoir of the Fil-ter Tube.•Centrifuge 15 s at 8,000 × g.•Remove the Filter Tube from the Collection Tube, discard theflowthrough liquid.•Combine the Filter Tube with the used Collection Tube.187.Quick Reference Procedure, continued¾•Add 500 ␮l Wash Buffer II (blue cap) to the upper reservoir of the FilterTube.•Centrifuge 15 s at 8,000 × g and discard the flowthrough.•Combine the Filter Tube with the used Collection Tube.µ•Add 300 ␮l Wash Buffer II (blue cap) to the upper reservoir of the FilterTube.•Centrifuge 2 min at full speed (approx 13, 000 × g).L The extra centrifugation time ensures removal of residual Wash Buf-fer.¸•Carefully remove the column from the collection tube so that the col-umn does not contact the flowthrough as this will result in carryover ofethanol.N Residual ethanol may interfere with subsequent reactions.•Insert the Filter Tube into a nuclease free, sterile 1.5 ml microcentri-fuge tube.¹•Add 100 ␮l Elution Buffer to the upper reservoir of the Filter Tube.•Centrifuge the tube assembly for 1 min at 8,000 × g.ƸThe microcentrifuge tube now contains the eluted RNA.L Either use 10 ␮l of the eluted RNA directly in RT-PCR or store theeluted RNA at -80°C for later analysis.。