罗氏第一链cDNA合成试剂盒Transcriptor First Strand cDNA Synthesis Kit 中文说明书
First Strand cDNA Synthesis Kit 说明书
GeneCopoeia Inc.19520 Amaranth DriveGermantown, Maryland 20874USATel: 301-515-6982; 1-866-360-9531Fax: 301-515-6983Web: First Strand cDNA Synthesis Kit产品套装编号：C0210A 储存条件：-20℃保存产品内容M-MLV Reverse Transcriptase (RNase H -)5×RT Reaction Buffer RNase Inhibitor 25mM dNTP60 µM Oligo(dT)18250 µM Random PrimerddH 2O (DNase/RNase Free)试剂组分total RNA 或Poly(A) mRNA60 µM Oligo(dT)18 或250 µM Random Primer 或10 µM Sequence-Speci fic Primer ddH 2O (DNase/RNase Free)产品编号C02010A C02011A C10200A C10011A C10210A C10220A C10230A体积1 µl 1 µl 1 µl包装规格50 μl (200 U/μl)250 μl50 μl (25 U/μl)50 μl 50 μl 50 μl 1 ml终浓度1 µg 10 ng 2.4 µM 10 µM 0.4 µM至总体积13 µl■ 产品概述：RNA 逆转录成的单链cDNA 是基因克隆和RNA 研究的主要材料。
本产品是采用莫洛尼鼠白血病病毒(Moloney Murine Leukemia Virus) 逆转录酶(M-MLV Reverse Transcriptase)来合成单链cDNA 的专用试剂盒。
ThermoScientificRevertAidFirstStrandcDNASynthesisKitK说明书第一链cDNA合成试剂盒
RevertAid第一链cDNA Synthesis试剂盒#K1621, #K1622分析证明书质量控制#K1621Lot采用100 fg对照GAPDH RNA和对照引物进行RT-PCR反应，通过在1%琼脂糖上进行凝胶电泳和溴化乙锭染色显示得到足够量的496 bp的产物质量认证人：Jurgita Zilinskiene目录页码试剂盒组成 (2)存储条件 (2)产品说明 (2)注意事项 (3)操作步骤 (6)RT-PCR (6)合成cDNA用于克隆………..………………………………………7 实验对照……………………………………………………………….8 问题分析与解决 (10)** 一个单位的RiboLockRNase酶抑制剂抑制5ng RNA酶A 50%的活性。
存储条件试剂盒中所有组分应存储在-20°C。
对照用RNA可存储于-70°C以便长期使用。
产品说明RevertAid第一链cDNA合成试剂盒以mRNA或者总RNA为模板，高效合成第一链cDNA。
本试剂盒使用RevertAid M-MuLV反转录酶，它的RNA酶H的活性与AMV反转录酶相比较低。
该反转录酶可耐受42-50°C温度，合成的cDNA片段长度达13kb。
试剂盒中含有RiboLock 重组RNA酶抑制剂，防止RNA降解，可耐受55°C高温。
试剂盒同时含有oligo(dT)18和随机六聚体引物。
随机六聚体引物与模板非特异性地结合，以总RNA中任何RNA为模板合成cDNA。
oligo(dT)18选择性和RNA 3’po ly(A)配对结合，只以有poly(A)尾巴的mRNA为模板合成cDNA。
使用本试剂盒也可采用序列特异性引物。
合成的第一链cDNA能直接用作PCR或荧光定量PCR的模板，第二链cDNA的合成或线性RNA扩增，也可用于需要用带有放射性或非放射性核苷酸标记第一链cDNA的实验，比如将标记好的第一链cDNA 作为杂交实验中的探针或者用于微阵列分析。
逆转录试剂盒( transcriptor first strand cdna synthesis kit ) 说明书
3
04 379 012 001
Transcriptor First Strand cDNA Synthesis Kit y Version 6.0
1. What this Product Does, continued
Vial/ Cap
Label
Store the kit at ؊15 to ؊25°C N Store Control RNA (vial 7 in
Cat. No 04 379 012 001) at —70°C or below.
y Version 6.0
Content version: September 2010
9 (7 for b,c) colorless
Water, PCR-grade
Content
a) Cat. No. 04 379 012 001 b) Cat. No. 04 896 866 001 c) Cat. No. 04 897 030 001
a) 1 vial, 1 ml b) 2 vials, each 1ml c) 3 vials, each 1 ml
L In Cat. No. 04 896 866 001 and Cat. No. 04 897 030 001 the control reagents (vial 7 and 8) are not included. Therefore, in these kits vial 7 is Water, PCR Grade.
Random Hexamer Primer
a) 1 vial, 100 l (600 M) b) 1 vial, 200 l (600 M) c) 2 vials, each 200 l (600 M)
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。
ThermoScientificMaxima第一链cDNA合成试剂盒适用于RT-qPCR
产品信息Thermo ScientificMaxima第一链cDNA合成试剂盒（适用于RT-qPCR）#K1641，#K1642/onebio分析证书经验证，在两步法RT-qPCR中，对于不同起始量（5 ng-0.5 fg）的RNA逆转录产物，使用Thermo Scientific Maxima SYBR Green/ROX qPCR 预混液进行qPCR扩增。
其反应效率在90-110％之间，曲线斜率在-3.09和-3.58之间，且相关系数>0.99。
质量授权人：Jurgita Zilinskiene目录页码试剂盒组分 (4)贮存条件 (4)产品描述 (4)重要提示 (5)RT-qPCR实验方案 (6)对照反应 (6)疑难解答 (7)参考文献 (8)试剂盒组分贮存条件所有试剂盒成分都应贮存于-20 °C。
产品描述Thermo Scientific Maxima第一链cDNA合成试剂盒是合成cDNA第一链的方便系统，适用于两步法实时定量RT-PCR（RT-qPCR）中的cDNA合成。
试剂盒采用先进的Maxima 反转录酶（RT），该酶源自M-MuL V 逆转录酶的体外进化。
这种酶相比M-MuL V逆转录酶热稳定性高，扩增效果强劲且具有更高的cDNA合成效率。
Maxima第一链cDNA合成试剂盒能够在较高温度下（55-65℃），在很宽的总RNA量范围内（从1 pg到5 μg）合成cDNA。
合成反应能在15-30分钟内完成。
Maxima第一链cDNA合成试剂盒的各组分经预混合，以节约时间和减少移液错误。
试剂盒由Maxima Enzyme Mix、5×反应混合液、无核酸酶的水三部分组成。
Maxima Enzyme Mix包含Maxima反转录酶和Thermo Scientific Ribolock RNase抑制剂。
重组的Ribolock RNase抑制剂能有效保护RNA模板在高于55℃时不被RNase A、B和C降解。
罗氏荧光定量说明书[1]
![罗氏荧光定量说明书[1]](https://img.taocdn.com/s3/m/2644ea79168884868762d65b.png)
Roche Applied ScienceFor general laboratory use. Not for use indiagnostic procedures. FOR IN VITRO USE ONLY.FastStart TaqMan ® Probe MasterFastStart TaqMan ®Probe Master (Rox)Cat. No. 04 673 409 001 2.5 ml (2 × 1.25 ml; 100 × 50 l reactions)Cat. No. 04 673 417 00112.5 ml (10 × 1.25 ml; 500 × 50 l reactions)Store at Ϫ15 to Ϫ25°CCat. No. 04 673 433 00150.0 ml (10 × 5 ml; 2000 × 50 l reactions)Cat. No. 04 673 450 001 2.5 ml (2 × 1.25 ml; 100 × 50 l reactions)Store at Ϫ15 to Ϫ25°C F Keep away from lightCat. No. 04 673 468 00112.5 ml (10 × 1.25 ml; 500 × 50 l reactions)Cat. No. 04 673 476 00150.0 ml (10 × 5 ml; 2000 × 50 l reactions)2× concentrated, ready-to-use hot start master mix for qPCR and qRT-PCR using the hydrolysis probe detection format on real-time PCR instruments (except the LightCycler ® Instruments)1.What this Product DoesContentsThe FastStart TaqMan ® Probe Master is a ready-to-use, 2× concen-trated master mix that contains all the reagents (except primers, probe and template) needed for running quantitative, real-time DNA detec-tion assays, including qPCR and 2-step qRT-PCR, in the hydrolysis probe detection format. It is available in two formulations, one that contains the Rox reference dye and one without Rox.Storage and StabilityIf stored at Ϫ15 to Ϫ25o C, the master mix is stable through the expira-tion date printed on the label. For short-term storage (up to 3months),the master mix may be stored at +2 to +8o C.N Keep the FastStart TaqMan ® Probe Master (Rox) away from light.N Avoid repeated freezing and thawing.L The complete PCR mix (i.e., FastStart TaqMan ® Probe Master sup-plemented with primers, probe, and template) is stable for up to 24hrs at room temperature. Keep the PCR mix away from light!ApplicationThe FastStart TaqMan ® Probe Master is a ready-to-use reagent mix that simplifies the preparation of reactions for qPCR and 2-step qRT-PCR. In combination with a real-time PCR instrument, suitable PCR primers and Hydrolysis Probe, FastStart TaqMan ® Probe Master allows very sensitive detection and quantification of defined DNA sequences.N Do not use this product on the LightCycler ® 1.5 Instrument, theLightCycler ® 2.0 Instrument, or the LightCycler ® 480 Instrument.In principle, the FastStart TaqMan ® Probe Master can be used for the amplification and detection of any DNA or cDNA target, including those that are GC-rich or GC-poor. However, you would need to adapt your detection protocol to the reaction conditions of the particular real-time PCR instrument in use and design a specific hydrolysis probe and PCR primers for each target. See the Operator’s Manual of your real-time PCR instrument for general recommendations.N The mix is designed for optimal amplification of targets up to500bp long. Do not use the mix to amplify longer targets.L FastStart TaqMan ® Probe Master offers convenience and ease-of-use because the addition of MgCl 2 to the reaction mixture is not necessary, thus avoiding time-consuming optimization steps.L The mix contains dUTP, so that it may be used with Uracil-DNAGlycosylase to prevent false positives arising from carry-over con-tamination, i.e., contamination with amplified DNA.L The FastStart TaqMan ® Probe Master is fully compatible withprobes from the Universal ProbeLibrary Sets*.Additional Equipment and Reagents RequiredAdditional reagents and equipment required to perform quantitative real-time PCR assays with the FastStart TaqMan ® Probe Master include:•general laboratory equipment–nuclease-free, aerosol-resistant pipette tips–pipettes with disposable, positive-displacement tips–sterile reaction tubes for preparing master mixes and dilu-tions–standard benchtop microcentrifuge –water, PCR-grade*•for first-strand cDNA synthesis–Transcriptor First Strand cDNA Synthesis Kit*•for real-time PCR–PCR reaction vessels (e.g., optical tubes or microplates)–sequence-specific primers–a hydrolysis probe (e.g ., from the Universal ProbeLibrary Sets*)–ROX Reference Dye* (optional)•for carry-over prevention (optional)–LightCycler ® Uracil-DNA Glycosylase** available from Roche Applied Science2.How to Use this Product2.1Before You BeginGeneralThe optimal reaction conditions (concentration of template DNA and PCR primers, incubation temperatures and times, cycle number)depend on the specific template/primer system and must be deter-mined individually.Sample Material•Use any template DNA (e.g., genomic or plasmid DNA, cDNA) suit-able for PCR in terms of purity, concentration, and absence of inhib-itors. For reproducible isolation of nucleic acids use:–either the MagNA Pure LC Instrument or the MagNA Pure Compact Instrument together with a dedicated nucleic acid isolation kit (for automated isolation) or–a HIGH PURE nucleic acid isolation kit (for manual isolation).0706.04699220001For details see the Roche Applied Science Biochemicals catalog or home page, .•Use up to 250 ng complex genomic DNA or 50 ng cDNA.PrimersUse PCR primers at a final concentration of 0.3 – 1.0 M. The recom-mended starting concentration is 0.9 M each.N Always use equimolar primer concentrations.N If you are using probes from the Universal ProbeLibrary*, use 200nM of each primer.L The design of the PCR primers determines amplicon length, melt-ing temperature, amplification efficiency, and yield. Primer design may also depend on the choice of PCR program (2-step versus 3-step protocol).L Several programs for primer design are freely available or provided by the suppliers of real-time PCR instruments (e.g., PrimerExpress).Alternatively, such programs are available to the public on the web for free. For example, use the free online ProbeFinder software () to design primers that may be paired with probes from the Universal ProbeLibrary*.ProbeThe probe concentration should be significantly lower than the primer concentration. As a starting point, we recommend using 250 nM probe. However, suitable concentrations range from 100 nM to 300nM.N To ensure a specific and sensitive assay, the probe must anneal to the DNA at a significantly higher temperature than the primers.Therefore, the T m of the probe should be 68 – 70°C and the T m of the primers about 58 – 60°C.N For maximum assay sensitivity, avoid placing a terminal G at the 5´-end of the probe because the fluorescent signal (arising after cleavage of the probe) is adversely affected by such a G.N To ensure that the fluorescent reporter dye within the unreacted probe is quenched, the length of the probe should not exceed 28 nucleotides.N If you use probes from the Universal Probe Library*, start with a probe concentration of 100 nM. Set the annealing temperature to 60°C.Negative ControlTo detect DNA contamination, always include a negative control in each run. To prepare this control, replace template DNA with PCR-grade water.Reaction VolumeVarious reaction volumes of the FastStart TaqMan® Probe Master can be used. Please refer to recommendations from the supplier of the instrument for suitable volumes and tubes/plates.ROX Reference DyeN Please read carefully.In principle, real-time PCR instruments (except the LightCycler®instruments) offer three different modes:1.Detection of released signal in relationship to a reference dye (usu-ally ROX)2.Detection of released signal in relationship to the quencher dye ofthe probe (e.g., TAMRA)3.Detection of released signal aloneThe choice of mode depends on the instrument (e.g. whether a chan-nel for detecting the reference dye is available) and on the light source of the instrument (halogen versus laser).The FastStart TaqMan® Probe Master is available with or without ROX. L The FastStart TaqMan® Probe Master (Rox) contains 120 nM ROX (2× concentrated), i.e. the final concentration of Rox in the PCR mix is 60 nM.The following list gives an overview how to apply the FastStart Taq-Man® Probe Master in combination with specific real-time PCR instru-ments:•If you want to use the reference channel of your real-time PCR instrument, then use the FastStart TaqMan® Probe Master (Rox).•for the ABI 7500 Real-Time PCR System, the Stratagene Mx3000P QPCR System, and the Corbett RotorGene Real Time Thermoana-lyzers: no addition of further ROX Reference Dye is required•for the ABI PRISM 7000 Sequence Detection System and the ABI7300 Real-Time PCR System: add additional ROX Reference Dyeto a final concentration of 300 nM a)•for the ABI 7700 Real-Time PCR System and the ABI PRISM7900HT Sequence Detection System: add additional ROX Refer-ence Dye to a final concentration of 400 nM a)•If you do not want to use the reference channel of your real-time PCR instrument or the instrument is not equipped with a reference channel, use either the FastStart TaqMan®Probe Master or the FastStart TaqMan® Probe Master (Rox).•If you use the Bio-Rad iCycler iQ5 Real-Time PCR Detection System apply only the External Well Factor Plate procedure for determining the well factors. For determining the well factors the Bio-Rad iCycler iQ5 Real-Time PCR Detection System does not use ROX but fluorescein. Therefore, use the FastStart TaqMan® Probe Mas-ter (without ROX) in combination with the iCycler iQ5 Real-Time PCR Detection System only! For details on how to perform the External Well Factor Plate procedure consult the iCycler iQ5 Real-Time PCR Detection System Instruction Manual.L a)This is the recommended starting concentration. The final con-centration of ROX Reference Dye may be further optimized in a range of 300 to 600 nM. Especially if you observe an unsteady flu-orescence signal, increase the ROX concentration until the signal is stable. Usually, when working with small reaction volumes (Յ10l) a higher ROX concentration is required. (In this case, the recommended starting concentration is 400 nM.)2.2ProcedurePreparation of the PCR mixFor each 50 l reaction, prepare the following reaction mix:ᕡ•Thaw the solutions and, for maximum recovery of the contents, briefly spin vials in a microcentrifuge before opening.•Mix solutions carefully by pipetting them up and down, thenstore on ice.ᕢPrepare 100× conc. solutions of the PCR primers and the hydrol-ysis probe.ᕣIn a 1.5 ml reaction tube on ice, prepare the PCR mix for one 50l reaction by adding the following components in the orderlisted below.Component Volume a)Finalconc.FastStart TaqMan® Probe MasterFastStart TaqMan® Probe Master (Rox)b25 l1×Hydrolysis Probe (25M)0.5 l250nMForward primer (90M)0.5 l900nMReverse primer (90M)0.5 l900nMWater, PCR-grade18.5 lT otal Volume45 lL a) To prepare the PCR mix for more than one reaction, multi-ply the amounts in the “Volume” column by z, where z = thenumber of reactions to be run + one additional reaction.N b) If you need to supplement the PCR mix with additional ROX Reference Dye*, add the dye to the FastStart TaqMan® ProbeMaster (Rox) before primers, probes or DNA template areadded. Refer to the package insert of the ROX Reference Dyefor detailed instructions.ᕤ•Mix the solution carefully by pipetting it up and down. Do not vortex.•Pipet45l PCR mix into each PCR reaction vessel or well of aPCR microplate (depending on your real-time PCR instrument).ᕥ•Add 5 l of template DNA (up to 250 ng total DNA) or cDNA.L In initial experiments to determine the optimum amount of cDNA template, we recommend running undiluted, 1:10diluted, and 1:100 diluted cDNA template in parallel.•Mix carefully by pipetting up and down.ᕦAccording to the instructions supplied with your instrument, prepare the tubes or microplates for PCR (e.g., seal tubes withoptical tube caps or the plate with self-adhesive foil).2Roche Applied Science3Roche Applied SciencePerforming PCRThere are several different ways to program the PCR. Either two-step or three-step PCR programs will provide suitable experimental results.The amplicon should be short (approx. 150 bp) and the annealing/elongation temperature should be 60°C (e.g., a typical PCR protocol is 40 cycles of 95°C/15 s, followed by 60°C/1 min).N For best results, be sure the instrument is calibrated correctly.2.3Related ProceduresPrevention of Carry-Over ContaminationUracil-DNA N-Glycosylase (UNG) is suitable for preventing carry-over contamination in PCR. This carry-over prevention technique involves incorporating deoxyuridine triphosphate into amplification products,then pretreating later PCR mixtures with UNG. If a dUTP-containing contaminant is present in the later PCRs, it will be cleaved by a combi-nation of the UNG and the high temperatures of the initial denatur-ation step; it will not serve as a PCR template. Since your target DNA template contains thymidine rather than uridine, it is not affected by this procedure.L dUTP is a component of the FastStart TaqMan ® Probe Master.N Perform prevention of carry-over contamination with LightCycler ®Uracil-DNA Glycosylase*. Add 1.25 U per 50 l PCR reaction. Pro-ceed as described in the package insert.Two-step RT-PCRFastStart TaqMan ® Probe Master can also be used to perform two-step RT-PCR. In two-step RT-PCR, the reverse transcription of RNA into cDNA is separated from the other reaction steps and is performed outside the real-time PCR instrument. Subsequent amplification and online monitoring is performed according to the standard real-time PCR procedure, using the cDNA as the starting sample material. Tran-scriptor First Strand cDNA Synthesis Kit* is recommended for reverse transcription of RNA into cDNA. Synthesis of cDNA is performed according to the detailed instructions provided with the kit.3.Troubleshooting³Following the instruction manual of the instrument supplier, program the instrument with the following parameters:CyclesAnalysis Mode Target Temperature Hold TimeRemarks 1 (optional)None 50°C2 minOnly if UNG hadbeen added for carry-over pre-vention 1None 95°C 10 minActivation of FastStart Taq DNA Polymerase 40Quantifi-cationDependent on the specific primer-probe combination.Amplification and real-time analysis·Place your tubes or plate in the instrument and start the reaction.»At the end of the reaction, follow instrument instructions for quantification/analysis.Problem CauseRecommendation No amplification detectable and no band in gel analysis•error in PCR program (e.g. activation step omitted)•pipetting errors (e.g. DNA not added)•amplicon too long •inhibitory effects of impurities•bad primer design•Adjust PCR program •Repeat experiment; check pipetting steps carefully•Redesign primer •Repeat isolation of template•Redesign primerNo or lowamplificationdetectable but strong band in gel analysisPCR is working but the probe is poorly designed Redesign probe 4.Additional Information on this ProductFastStart Taq DNA PolymeraseThe FastStart TaqMan ® Probe Master (with or without ROX) contains the FastStart Taq DNA Polymerase for hot-start PCR to improve speci-ficity and sensitivity of the PCR by minimizing the formation of non-specific amplification products (1,2,3,4). This enzyme delivers superior results thanks to its unique enzyme design and optimized buffer sys-tem.FastStart Taq DNA Polymerase is a chemically modified form of ther-mostable recombinant Taq DNA polymerase that shows no activity up to 75°C. The enzyme is active only at high temperatures, where primers no longer bind non-specifically. The enzyme is completely activated (by removal of blocking groups) in a single pre-incubation step (95°C,10 min) before cycling begins. Activation does not require the extra handling steps typical of other hot-start techniques.Detection of PCR productsReal-time DNA detection assays based on the hydrolysis probe format (also known as 5´-nuclease assays) require a single, signal-generating probe that contains two labels, a fluorescent reporter dye at the 5´-end and a (fluorescent or dark) quencher label at or near the 3´-end (6).When the probe is intact, the fluorescent signal is almost completely suppressed by the quenching label. When the probe is hybridized to its target sequence, it is cleaved by the 5´→3´ exonuclease activity of the FastStart Taq DNA Polymerase, which “unquenches” the fluores-cent reporter dye. During each PCR cycle, more of the released fluo-rescent dye accumulates, boosting the fluorescent signal.N If you use the hydrolysis probe format for detection, you cannotperform a subsequent melting curve analysis. For melting curve analysis, we recommend using the FastStart SYBR Green Master*.Quality ControlEach lot is tested for performance in qPCR using three templates:a GC–rich template, a GC-poor template and a long template (about 440 bp).References1Chou, Q et al (1992). Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nuc. Acid Res. 20, 1717-1723.2Kellogg, DE et al (1994). TaqStart Antibody: hot-start PCR facili-tated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16, 1134-1137.3Birch, DE et al (1996). Simplified hot start PCR. Nature 381, 445-446.4PCR Manual, Roche Diagnostics (1999) 2nd edition (1999) 2, 52-58.5Bustin, SA (ed ., 2004). A – Z of Quantitative PCR. IUL Biotech-nology Series, 5.6Holland, PM et al (1991). Detection of specific polymerase chain reaction product by utilizing the 5´→3´ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci . USA . 88, 7276-7280.7Rees, E. et al (2006). siRNA Silencing of Lamin A and Quantifi-cation of the Knockdown Effect via qPCR. Biochemica 2006 (2), 25-27.Fluorescence varies within a run instrument not correctly calibratedRecalibrate instrument variations in pipettingMonitor the channel in which Rox is detected High background in the negative (no template) controlContamination •Remake or replace critical solutions (e.g ., water)•Clean lab bench •Use UNG to prevent carry-over contami-nationProblem Cause RecommendationRoche Diagnostics GmbHRoche Applied Science 68298 Mannheim Germany5.Supplementary Information5.1ConventionsText ConventionsTo make information consistent and memorable, the following text conventions are used in this package insert:SymbolsIn this package insert the following symbols are used to highlight important information:5.2AbbreviationsIn this Instruction Manual, the following abbreviations are used:5.3Changes to Previous Version•Storage and Stability information extended.•Recommended concentrations of ROX for ABI real-time PCR instruments changed.•Minor editorial changes5.4Ordering 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 book-mark our home page, , and our Special Interest Sites including:•The Universal ProbeLibrary: •The MagNA Pure System family for automated nucleic acid isolation: •Amplification – Innovative Tools for PCR: /pcr•DNA & RNA preparation – Versatile Tools for Nucleic Acid Purification: /napure L Refills for all 165 individual Universal ProbeLibrary probes, each sufficient for 500(50 l) reactions, can be ordered at .Text Convention UseNumbered Instructions labeled ቢ, ባ,etc.Steps in a process that usually occur in the order listed Numbered Instructions labeled ³, ·,etc.Steps in a procedure that must be performed in the order listed Asterisk *Denotes a product available from Roche Applied ScienceSymbolDescriptionL 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.Abbreviation MeaningqPCR quantitative real-time PCR UNGUracil-DNA N-GlycosylaseProductPack SizeCat. No.FastStart SYBR Green Master (Rox) 5 ml (4 × 1.25 ml)50 ml (10 × 5 ml)04 673 514 00104 673 522 001FastStart SYBR Green Master 5 ml (4 × 1.25 ml)50 ml (10 × 5 ml)04 673 484 00104 673 492 001ROX Reference Dye50 l (1 mM)04 673 549 001Transcriptor First Strand cDNA Syn-thesis Kit1 kit 04 379 012 001LightCycler ® Uracil-DNA Glycosy-lase100 U 03 539 806 001Water, PCR Grade 25 ml (25 × 1 ml)25 ml (1 × 25 ml)100 ml (4 × 25 ml)03 315 932 00103 315 959 00103 315 843 001High Pure PCR T emplate Preparation Kit100 purifications 11 796 828 001mRNA Isolation Kit Ͼ70 g mRNA11 741 985 001Universal ProbeLibrary Set, Arabi-dopsis Library of 90 pre-validated detection probes04 683 595 001Universal ProbeLibrary Set, C. ele-gans Library of 90 pre-validated detection probes04 683 609 001Universal ProbeLibrary Set, Droso-phila Library of 90 pre-validated detection probes04 683 625 001Universal ProbeLibrary Set, Human Library of 90 pre-validateddetection probes04 683 633 001Universal ProbeLibrary Set, Mouse Library of 90 pre-validateddetection probes04 683 641 001Universal ProbeLibrary Set, Primates Library of 90 pre-validateddetection probes04 683 617 001Universal ProbeLibrary Set,Rat Library of 90 pre-validated detection probes04 683 650 001NOTICE TO PURCHASERLIMITED LICENSE: A license to perform the 5' nuclease process for research requires the use of a Licensed 5' Nuclease Kit (containing Licensed Probe), or the combination of an Authorized Core Kit plus Licensed Probe, or license rights that may be purchased from Applied Biosystems. This product is an Authorized Core Kit without Licensed Probe. Its purchase price includes a limited, non-transferable immu-nity from suit under U.S. Patents Nos. 5,210,015, 5,487,972, 5,476,774,and 5,219,727, and corresponding patent claims outside the United States, owned by Roche Molecular Systems, Inc. or F. Hoffmann-La Roche Ltd ("Roche"), for using only this amount of the product in the practice of the 5' nuclease process solely for the purchaser's own internal research and development activities. This product is also an Authorized Core Kit for use with service sublicenses available from Applied Biosystems. This product conveys no rights under U.S. Pat-ents Nos. 5,804,375, 6,214,979, 5,538,848, 5,723,591, 5,876,930,6,030,787, or 6,258,569, or corresponding patent claims outside the United States, expressly, by implication, or by estoppel.No right under any other patent claims (such as apparatus or system claims in U.S. Patent No. 6,814,934) and no right to perform commer-cial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consider-ation, is hereby granted expressly, by implication, or by estoppel. This product is for research purposes only. Diagnostic uses require a sepa-rate license from Roche.Further information on purchasing licenses to practice real-time PCR processes may be obtained by contacting the Director of Licensing,Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.TrademarksLIGHTCYCLER, FASTSTART, TAQMAN, HYBPROBE, MAGNA PURE,and HIGH PURE are Trademarks of Roche.ROX, PRISM, TAMRA, and PRIMER EXPRESS are Trademarks of Applera Corporation, Norwalk, CT, USA.Other brand or product names are trademarks of their respective holders.Contact and SupportTo ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at:/supportTo call, write, fax, or email us, visit the Roche Applied Science home page, , and select your home country. Country-specific contact information will be displayed.Use the Product Search function to find Pack Inserts and Material Safety Data Sheets.。
ThermoScientificMaxima第一链cDNA合成试剂盒适用于RT-qPCR
产品信息Thermo ScientificMaxima第一链cDNA合成试剂盒（适用于RT-qPCR）#K1641，#K1642/onebio分析证书经验证，在两步法RT-qPCR中，对于不同起始量（5 ng-0.5 fg）的RNA逆转录产物，使用Thermo Scientific Maxima SYBR Green/ROX qPCR 预混液进行qPCR扩增。
其反应效率在90-110％之间，曲线斜率在-3.09和-3.58之间，且相关系数>0.99。
质量授权人：Jurgita Zilinskiene目录页码试剂盒组分 (4)贮存条件 (4)产品描述 (4)重要提示 (5)RT-qPCR实验方案 (6)对照反应 (6)疑难解答 (7)参考文献 (8)试剂盒组分贮存条件所有试剂盒成分都应贮存于-20 °C。
产品描述Thermo Scientific Maxima第一链cDNA合成试剂盒是合成cDNA第一链的方便系统，适用于两步法实时定量RT-PCR（RT-qPCR）中的cDNA合成。
试剂盒采用先进的Maxima 反转录酶（RT），该酶源自M-MuL V 逆转录酶的体外进化。
这种酶相比M-MuL V逆转录酶热稳定性高，扩增效果强劲且具有更高的cDNA合成效率。
Maxima第一链cDNA合成试剂盒能够在较高温度下（55-65℃），在很宽的总RNA量范围内（从1 pg到5 μg）合成cDNA。
合成反应能在15-30分钟内完成。
Maxima第一链cDNA合成试剂盒的各组分经预混合，以节约时间和减少移液错误。
试剂盒由Maxima Enzyme Mix、5×反应混合液、无核酸酶的水三部分组成。
Maxima Enzyme Mix包含Maxima反转录酶和Thermo Scientific Ribolock RNase抑制剂。
重组的Ribolock RNase抑制剂能有效保护RNA模板在高于55℃时不被RNase A、B和C降解。
cdna_synthesis_kit
0.2-2 μg 1 μl 1 μl 4在PCR仪上按以下条件进行反转录反应:
30°C
5min
42°C 30-40min
95°C
5min
4°C
产品组分
组分名称 M-MuLV 反转录酶 (200 U/μl) 5XRT PCR Buffer dNTP Mixture (10 mM each) Rnase Inhibitor (40 U/μl) Oligo (dT)15 Primer (20 μM)
cDNA 第一链合成试剂盒 1st cDNA Synthesis Kit
使用说明书
Cat. No.: D0401 包装量：50次
产品说明
本试剂盒采用莫洛尼氏鼠白血病病毒反转录酶M-MuLV ，该酶通过点突变使RNase H活性缺失，而保持完全的 mRNA-directed DNA多聚合活性，有效增强了cDNA的 合成效率、增加了该酶与模板/引物的亲和力, 从而增 加了酶的活性和持续合成能力。本试剂盒优化了反应 Buffer，能使反应的模板量（Total RNA）可在0.2-2 μg 范围之间进行逆转录反应。cDNA合成后进行PCR扩增 既可检测到长cDNA（>10 kb）片段，也可检测到低丰 度cDNA样品。本试剂盒提供了由Total RNA或mRNA合 成cDNA第一链所需要的全部试剂，并提供两种cDNA合 成引物。合成的第一链cDNA可广泛用于2nd Strand的合 成、杂交、PCR扩增、Real-Time PCR反应等。
Rnase Free H2O
储存条件
-20°C保存，避免反复冻融。
数量 50 μl 250 μl 50 μl 25 μl 50 μl
1 ml
操作方法
1．按以下组分配制反转录反应液
Thermo Scientific Rever id First Strand cDNA Synthesis Kit K 说明书 第一链cDNA合成试剂盒
#K1621, #K1622
分析证明书 #K1621 Lot
质量控制
采用100 fg对照GAPDH RNA和对照引物进行RT-PCR反应，通过在1%琼脂糖上进行凝胶电泳和溴 化乙锭染色显示得到足够量的496 bp的产物
质量认证人：Jurgita Zilinskiene
RT-PCR…………………………………………………………….6 合成cDNA用于克隆………..………………………………………7 实验对照……………………………………………………………….8 问题分析与解决……………………………………………………...10
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试剂盒成分
RevertAid™第一链cDNA合成试剂盒
2. 37°C 孵育 30 分钟。
3. 加入1 μl 50 mM EDTA，65°C 孵育10分钟。在含有二价阳离子(1)而缺乏螯合剂的环境中，RNA
产品说明
RevertAid™第一链cDNA合成试剂盒以mRNA或者总RNA为模板，高效合成第一链cDNA。本试剂 盒使用RevertAid™ M-MuLV反转录酶，它的RNA酶H的活性与AMV反转录酶相比较低。该反转录酶可 耐受42-50°C温度，合成的cDNA片段长度达13kb。
试剂盒中含有RiboLock™ 重组RNA酶抑制剂，防止RNA降解，可耐受55°C高温。 试剂盒同时含有oligo(dT)18和随机六聚体引物。随机六聚体引物与模板非特异性地结合，以总RNA 中任何RNA为模板合成cDNA。oligo(dT)18选择性和RNA 3’poly(A)配对结合，只以有poly(A)尾巴的 mRNA为模板合成cDNA。使用本试剂盒也可采用序列特异性引物。 合成的第一链cDNA能直接用作PCR或荧光定量PCR的模板，第二链cDNA的合成或线性RNA扩增， 也可用于需要用带有放射性或非放射性核苷酸标记第一链cDNA的实验，比如将标记好的第一链cDNA 作为杂交实验中的探针或者用于微阵列分析。
BeyoRT cDNA第一链合成试剂盒(RNase H-) 说明书
BeyoRT cDNA第一链合成试剂盒(RNase H-)产品编号产品名称包装cDNA第一链合成试剂盒(RNase H-) 10次D7166 BeyoRT产品简介：¾BeyoRT cDNA第一链合成试剂盒(RNase H-)，即BeyoRT First Strand cDNA Synthesis Kit (RNase H minus)是一种采用了经过改造和优化的BeyoRT M-MuLV反转录酶(RNase H-)，用于在总RNA、mRNA等RNA模板的基础上反转录产生cDNA第一链的试剂盒。
本试剂盒包含了进行cDNA第一链合成所需的各种试剂。
¾采用本试剂盒可以合成长达13kb的cDNA第一链。
在采用BeyoRT M-MuLV反转录酶(RNase H-)的情况下，由于缺失了RNase H，RNA和DNA双链复合物中的RNA不会被降解，这样反转录出来的cDNA的长度就会更长，并且产量更高。
¾试剂盒中提供了RNase Inhibitor，确保在反转录过程中的RNA不会被RNA酶所降解并取得较好的反转录效果。
¾试剂盒还提供了Oligo(dT)18和random hexamer这两种引物，前者适合用于带有Poly(A)尾的mRNA的反转录，后者进行反转录时不需要poly(A)尾。
也可以自行采用基因特异性引物进行cDNA第一链的合成。
¾试剂盒中提供的Control Primer为17聚，即引物的长度为17个碱基。
试剂盒中提供的Control RNA为带有3’ poly(A)的1.1kb RNA。
¾使用本试剂盒合成的第一链，可以直接用于后续的常规PCR、real-time PCR、cDNA第二条链的合成等。
¾本试剂盒用于体积为20微升的cDNA第一链合成反应时足够进行10个cDNA第一链样品的合成。
包装清单：产品编号产品名称包装D7166-1 BeyoRT M-MuLV反转录酶(RNase H-) 2000UD7166-2 Reaction Buffer (5X) 50µlD7166-3 RNase Inhibitor (20U/µl) 10µlD7166-4 dNTP Mix (10 mM each) 20µlD7166-5 Oligo(dT)18 Primer (0.5µg/µl) 10µlD7166-6 Random Hexamer Primer (0.2µg/µl) 10µlD7166-7 Control RNA (20ng/µl) 6µlD7166-8 Control Primer (5pmol/µl) 6µlD7166-9 DEPC-treated Water 0.2ml－说明书1份保存条件：-20℃保存。
逆转录试剂盒( transcriptor first strand cdna synthesis kit )操作步骤
Transcriptor First Strand cDNA Synthesis Kit 逆转录步骤1，使用前解冻结冰的试剂2，将试剂短暂离心3，冰上操作：（操作RNA实验需要戴手套）将模板与引物在PCR管上混合试剂体积最终浓度细胞总RNA 或 1 ug 总RNA 或10ng多聚尾(A)+m RNA 多聚尾(A)+ mRNA任选其中一个引物:Anchored-oligo(dT)18 Primer, 1 ul 2.5 uM50 pmol/ul (vial 5)或Random Hexamer Primer, 2ul 60uM600 pmol/ul (vial 6)或 Sequence-Specific Primer 可变0.5-2.5 uM1%DEPC水, (vials 7 or 9) 可变使总体积为 13 ul总体积13ul注：以上为初次实验的推荐浓度。
总RNA的适用浓度为10ug到5ng，以及mRNA的适用浓度为1到100ng,当RNA模板浓度较低时添加10ug/ml 的MS2 RNA* 来稳定模板RNA。
4，（可选）65度水浴上述混合物10分钟，使模板引物混合物变性（确保失活RNA二级结构），水浴后立即冰浴至少一分钟5，往上述混合物继续加入以下试剂（冰上操作）试剂体积最终浓度.Transcriptor Reverse Transcriptase 4 ul 1×(8 mM MgCl2) Reaction Buffer, 5× conc. (vial 2)Protector RNase Inhibitor, 0.5 ul40 U/ul (vial 3)20 U Deoxynucleotide Mix, 2 ul 每个1 mM 10 mM each (vial 4)Transcriptor Reverse 0.5 ul 10U Transcriptase, 20 U/ul (vial 1)最终体积20 ul注：小心混合上述试剂，不要振荡，短暂离心使试剂沉在管底6，将PCR管放在PCR仪上孵育，孵育条件如下：使用的引物目标mRNA长度孵育温度与时间Anchored-oligo(dT)18 不大于4kb 55度30分钟大于4kb 50度60分钟primer, 50 pmol/ul 或Sequence–specific primerRandom hexamer primers, 不大于4kb 25度10分钟，然后55度30分钟600pmol/ul 大于4kB 25度10分钟，然后50度60分钟6,85℃水浴5分钟灭活逆转录酶8、－20℃保存，或立即进行PCR。
Foregene RT EasyTM I 说明书
RT Easy TM IMaster Premix for first-strand cDNA synthesisCat.No.RT-01011/01012Fast and highly sensitive reverse transcription system for generating first-strand cDNA using pg-level RNAFor research use only Store at-20℃目录产品介绍 (3)产品特点 (3)试剂盒应用 (3)产品质量控制 (4)试剂盒内容 (4)运输及储存条件 (4)试剂盒组分信息 (5)注意事项 (5)RT Mix耐受性 (6)●酒精耐受性分析 (6)●胍盐耐受性分析 (6)操作前准备事项 (7)逆转录引物用量 (7)RNA模板用量 (7)实验材料和设备 (7)自备试剂 (8)安全性 (8)操作指南 (9)操作示意图 (10)问题分析指南 (11)本公司的2×RT Easy TM Mix合成第一链cDNA的温度高达50℃，有利于复杂二级结构RNA模板进行逆转录反应。
实验室纯化获得的RNA常有酒精及胍盐残留，对大多数逆转录酶有很强的抑制性，导致逆转录效果不理想或逆转录效率低。
福际2×RT Easy TM Mix对酒精和胍盐显示出极高的耐受性，对RNA样品中酒精的最高耐受为45%，胍盐的最高耐受为750mM。
即使不纯的RNA也可用福际2×RT Mix进行逆转录反应。
独特的反应体系,使得RT反应更加简便、快捷、高效，25min即可完成第一链cDNA的合成。
此产品可与Foregene公司的荧光定量产品Real Time PCR Easy TM配合使用，能获得高质量的实验结果。
产品特点◆高效的逆转录体系，只需25min即可完成第一链cDNA的合成。
◆高灵敏的逆转录体系，pg级别的模板也可以得到高质量的cDNA。
Roche荧光定量试剂盒Fast...
Roche荧光定量试剂盒Fast...注意：该使用指南为简易版，在实际使用时请查阅正式英文说明书。
1. 产品概述产品描述FastStart Universal SYBR Green Master(ROX)为即用型、2×浓度的反应混和液，该产品包含了基于SYBR Green I检测模式的qPCR 和两步法qRT-PCR进行实时DNA检测时所需的所有试剂(除了引物与模板)。
该试剂内含参比染料ROX，适用于所有需要ROX校正的实时荧光定量PCR平台。
储存条件及其稳定性该产品在-15~-25°C条件下可稳定存放至标签上的有效期之前。
若存放于+2~+8°C条件下，则可短期储存（不超过1个月）。
注意：请将该产品避光保存。
请避免将该产品反复冻融。
该反应液添加引物与模板后可在+15~+25°C及避光条件下稳定储存24小时。
该产品用干冰运输。
2. 产品使用方法2.1 实验前准备实验最佳反应条件(包括DNA模板和PCR引物浓度、孵育温度和时间、循环次数)应根据特异性模板/引物而异。
样本准备•请使用纯度、浓度经优化摸索且不含PCR抑制物的DNA模板(如，基因组学或质粒DNA，cDNA)。
为获得高重复性的核酸分离产物，请使用以下试剂：- MagNA Pure LC或MagNA Pure Compact系统结合系统配套的核酸分离试剂盒用于核酸自动分离。
- 手动分离请使用High Pure Nucleic Acid Isolation Kit。
更多详细信息，请阅读罗氏应用科学部生化产品目录，或访问罗氏应用科学部主页•请使用至多250 ng基因组DNA或50 ng cDNA。
请将DNA模板储存于PCR级别的水中或5 –10 mM 的Tris/HCl(pH 7.5-8.0)中。
由于EDTA与镁离子会形成敖合物，故请避免将模板溶解于TE缓冲液。
引物PCR引物的最终浓度为0.1 –0.4 μM。
反转录RT-PCR的相关事项
1．Money 鼠白血病病毒（MMLV）反转录酶：有强的聚合酶活性，RNA酶H活性相对较弱。
最适作用温度为37℃。
2．禽成髓细胞瘤病毒（AMV）反转录酶：有强的聚合酶活性和RNA酶H活性。
最适作用温度为42℃。
3．Thermus thermophilus、Thermus flavus等嗜热微生物的热稳定性反转录酶：在Mn2 存在下，允许高温反转录RNA，以消除RNA模板的二级结构。
4．MMLV反转录酶的RNase H-突变体：商品名为SuperScript 和SuperScriptⅡ。
此种酶较其它酶能多将更大部分的RNA转换成cDNA，这一特性允许从含二级结构的、低温反转录很困难的mRNA模板合成较长cDNA。
二、合成cDNA引物的选择1．随机六聚体引物：当特定mRNA由于含有使反转录酶终止的序列而难于拷贝其全长序列时，可采用随机六聚体引物这一不特异的引物来拷贝全长mRNA。
用此种方法时，体系中所有RNA分子全部充当了cDNA第一链模板，PCR 引物在扩增过程中赋予所需要的特异性。
通常用此引物合成的cDNA中96%来源于rRNA。
2．Oligo(dT)：是一种对mRNA特异的方法。
因绝大多数真核细胞mRNA具有3’端Poly(A )尾，此引物与其配对，仅mRNA可被转录。
由于Poly(A )RNA仅占总RNA的1-4%，故此种引物合成的cDNA比随机六聚体作为引物和得到的cDNA在数量和复杂性方面均要小。
3．特异性引物：最特异的引发方法是用含目标RNA的互补序列的寡核苷酸作为引物，若PCR反应用二种特异性引物，第一条链的合成可由与mRN A 3’端最靠近的配对引物起始。
用此类引物仅产生所需要的cDNA，导致更为特异的PCR扩增。
三、操作步骤1. 总RNA的提取。
2. cDNA第一链的合成（Reverse Transcription）：目前试剂公司有多种cDNA第一链试剂盒出售，其原理基本相同，但操作步骤不一。
cDNA第一链合成试剂盒
cDNA第一链合成试剂盒操作方法1、逆转录反应（1）在无菌薄壁管中加入以下成份：Total RNA 0.5-1.0µg(dN)9 or oligo(dT)18100pmol（2）混匀后，70℃变性5min，将薄壁管迅速置于冰上冷却，然后依次加入以下成份：5×M-MLV Buffer 4µldNTPs（10mM each）1µlRNasin 10U100mM DTT 2µlM-MLV Reverse Transcriptase 100UDEPC-treated water up to 20µl（3）混匀后短暂离心，使用oligo(dT)18引物时在42℃，使用随机引物(dN)9时在37℃下温育1小时。
（4） 94℃ 5min终止反应后，冰上冷却。
（5）合成的cDNA第一链可直接用于PCR扩增或进行其它下游操作。
2、P CR扩增（1）在PCR薄壁管中加入以下试剂Synthesized cDNA 2-5µl10×PCR Buffer 2µlUpper primer 10pmolLower primer 10pmoldNTPs（10mM each）0.5µlTaq DNA Polymerase 1.5UddH2O up to 20µl（2）混匀后短暂离心，然后进行PCR扩增。
94℃预变性2.5min。
进入下列30~40个循环（此扩增条件仅供参考）：94℃30s，60℃ 45s，72℃ 1min~3min。
最后72℃延伸7min。
注意事项1、确保RNA完整性及纯度。
RNA质量是决定合成cDNA第一链成功的关键因素，其纯度及完整性可由OD260/OD280比值和进行琼脂糖凝胶电泳检测。
较纯的RNA样品OD260/OD280比值通常为1.8-2.0，若该比值偏低，应进一步纯化。
真核生物RNA样品电泳图谱28s和18s的比值约为2:1，否则，表明有RNA降解。
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反应次数目录号反应次数04 379 012 00150次，包括10次对照反应04 896 866 001100次04 897 030 001200次试剂盒包装与含量小瓶/瓶盖标签适用于a) 04 379 012 001 b) 04 896 866 001 c) 04 897 030 0011红色TranscriptorReverseTranscriptase（逆转录酶）a) 1瓶，25 μl (20 U/μl)b) 1瓶，50 μl (20 U/μl)c) 2瓶，各50 μl (20 U/ μl)储存缓冲液：200 mM 磷酸钾，2 mM 二硫苏糖醇，0.2% Triton X-100(v/v)，50% 甘油(v/v)，pH 约为7.2∙2无色Transcriptor RTReaction Buffer(5×)（逆转录缓冲液）a) 1瓶，1 mlb) 1瓶，1 mlc) 2瓶，各1 ml5×浓度：250 mM Tris/HCl，150 mM KCl，40 mM MgCl2，pH约为8.5(25°C)∙3无色ProtectorRNase Inhibitor（RNase抑制剂）a) 1瓶，50 μl(40 U/μl)b) 1瓶，100 μl(40 U/μl)c) 2瓶，各100 μl(40 U/μl)储存缓冲液：20 mM Hepes-KOH，50 mM KCl，8 mM 二硫苏糖醇，50 % 甘油(v/v)，pH 约为7.6 (4°C)∙4黄色/ 紫色Deoxynuc-leo-tideMix（dNTP）a) 1瓶，100 μl(黄色瓶盖)b) 1瓶，200 μl(紫色瓶盖)c) 2瓶，各200 μl(紫色瓶盖)dATP, dCTP, dGTP, dTTP各10 mM∙5蓝色Anchored-oligo(dT)18Primer（锚定oligo(dT)18引物）a) 1瓶，100 μl(50 μM)b) 1瓶，200 μl(50 μM)c) 2瓶，各200 μl(50 μM)6Random Hexamera) 1瓶，100 μl(600 μM)蓝色Primer（随机引物）b) 1瓶，200 μl(600 μM)c) 2瓶，各200 μl(600 μM)7绿色Control RNA（对照RNA）a) 1瓶，20 μl(50 ng/μl)包含提取于永生细胞系(K562)的总RNA 片段稳定溶液∙8绿色Control Primer Mix PBGD（对照基因引物）a) 1瓶，40 μl5 μM 人类PBGD特异性正向与反向引物∙9(b和c为瓶7)无色Water, PCR-gradea) 1瓶，1 mlb) 2瓶，各1 mlc) 3瓶，各1 ml注意：货号为04 896 866 001和04 897030 001的产品不含有control试剂(瓶7和瓶8)，因此瓶7即为Water, PCR-grade。
储存条件及其稳定性请将该产品存放于-15~-25°C条件中，并于产品标签上的有效期之前使用。
注意：Control RNA(货号04 379 012 001的瓶7)应储存于-70°C条件。
请避免将产品反复冻融！2 产品使用方法2.1 实验前准备样本材料RNA模板：分离的总RNA，mRNA，病毒RNA或体外转录的RNA。
注意：想要得到较好的RT结果，使用高质量完整的RNA(不含基因组DNA残留、RNA酶、抑制剂)是必要的。
请根据以下预防措施进行操作，避免使RNA在分离过程中受到RNA酶的污染(从细胞裂解开始)：- 使用RNA酶抑制剂(如Protector RNase Inhibitor)或在使RNA酶失活的分离条件下进行操作。
- 如果有必要，用凝胶电泳分析过程中不同步骤(裂解、分离)以确保样本中不含有RNA酶。
- 请记住RNA酶也可能出现在受到污染的玻璃容器中。
为了准备总RNA或mRNA，我们建议您使用罗氏应用科学部的试剂。
为了筛选出能够产生高质量的适用于RT-PCR的完整RNA模板，请参考第3部分。
补充信息：想了解订货信息或查看相关核酸分离纯化指南，请访问/napure.想了解关于使用MagNA Pure LCSystem或MagNA Pure Compact System进行核酸自动分离的更多信息，请访问.引物六聚物随机引物∙在常规的RT反应中，使用5μg的总RNA 模板，可采用60μM的随机引物终浓度。
使用5μg总RNA并增加随机引物浓度，将增加短片段PCR产物（的产率，但是相应地，长片段PCR产物及全长转录子的产率会降低。
在非特异引物的反转录方法中，随机引物法是最多使用的一种方法，其特异性的扩增产物只能通过后续的PCR反应中使用特异的PCR引物获得。
锚定oligo(dT)18引物∙锚定oligo(dT)18引物特异性地结合在带有poly(A)+的RNA片段上，这类片段通常只占总RNA的1-2%，因此用锚定oligo(dT)18引物进行反转录所获得的cDNA产率大大低于随机引物方法。
如果实验是为了获得新mRNA的RT-PCR产物，则推荐使用锚定oligo(dT)18引物。
使用该方法获得的RT-PCR产物具有较强的一致性。
特异性序列引物∙使用特异性序列引物，其终浓度推荐为2μM。
但是请注意，即使这些引物在以DNA作为模板的PCR反应中成功获得扩增产物，相同的引物序列却可能难以定位在cDNA片段上。
如果遇到此类情况，请使用锚定oligo(dT)18引物重新进行第一链合成反应。
2.2 实验流程标准RT-PCR流程在此提供两种不同的操作流程：A：反转录使用锚定oligo(dT)18引物或六聚物随机引物或特异性序列引物。
在大多数情况下，只选择其中的一种引物进行cDNA合成。
∙B：反转录使用oligo(dT)18引物和六聚物随机引物的混合物。
这种方法可提高反应敏感度，但与使用单一oligo(dT)18引物比起来反应特异性会降低。
∙注意：根据您使用的引物类型，参考以下给出的流程A与B。
流程A：使用锚定oligo(dT)18引物或六聚物随机引物或特异性序列引物合成cDNA。
以下描述的条件适用于两步法RT-PCR的第一链cDNA合成。
图1为单一反应和多重反应中cDNA合成流程概述。
单一反应多重反应RNA+引物+水可选：变性10min 65°C冰上配置多重RT反应混合液(除RNA模板)并分装到各反应管添加模板RNA到每个反应管中置于冰上，添加缓冲液，RNase Inhibitor，dNTPs，Transcriptor RT锚定oligo(dT)18引物/特异性序列引物30min 55°C或1h 50°C六聚物随机引物10min 25°C30min 55°C或1h 50°C85°C失活5min添加1-5 μl至PCR体系或RT-PCR体系准备cDNA合成图1：单一反应和多重反应中cDNA合成流程概述①使用前先将所有冷冻试剂解冻。
∙开始实验前将所有试剂轻度离心。
∙实验开始后保持将所有试剂置于冰上。
∙②将无菌、无核酸酶、薄管壁的PCR管置于冰上，以20 μl反应体系为例，按照以下组分准备模板-引物混合物。
注意：进行与RNA相关的操作时请保持配戴手套。
模板-引物混合物(1次反应) 成分体积最终浓度总RNA或poly(A)+ mRNA1 μg 总RNA或10 ng poly(A)+ mRNA引物- 以下任选其一：Anchored-oligo(dT)18 Primer, 50pmol/μl (瓶5)1 μl2.5 μM 或Random Hexamer Primer, 600 pmol/μl (瓶6)2 μl60 μM或Sequence-Specific Primer 可变0.5-2.5 μMWater, PCR-grade (瓶7或9)可变使总体积为13 μl总体积13 μl以上为初实验的建议浓度。
为了得到合适的模板浓度，需要使总RNA的量在10 ng 到5 μg之间，使mRNA的量在1 ng到100ng之间。
注意：当RNA样本浓度较低(< 10 μg/ml)时，添加10 μg/ml MS2 RNA*以使RNA 模板稳定。
③可选步骤：在65°C条件下加热反应管10min使模板-引物混合物变性。
此步骤可确保RNA 二级结构变性。
∙立即将反应管置于冰上冷却。
∙④在含有模板-引物混合物的反应管中加入下表中其余的RT反应液组分。
成分体积最终浓度Transcriptor Reverse Transcriptase Reaction Buffer, 5×(瓶2)4 μl1×(8 mM MgCl2)Protector RNase Inhibitor,40 U/μl(瓶3)0.5 μl20 UDeoxynucleotide Mix,每种dNTP各10 mM(瓶4)2 μl每种dNTP各1 mMTranscriptor Reverse Transcriptase,20 U/μl(瓶1)0.5 μl10 U最终体积20 μl⑤在反应管中将试剂小心混匀。
∙注意：不要旋涡！将反应管轻度离心使样本收集于反应管底部。
∙反应时使用带热盖的加热模块，防止反应管中的液体挥发。
∙⑥根据您所使用的引物以及目标mRNA的长度，RT反应孵育时间参考下表：使用的引物目标mRNA的长度RT反应孵育时间Anchored-oligo(dT)18 Primer, 50pmol/μl 或Sequence-Specific Primer不超过4 kb55°C下30min超过4 kb50°C下60minRandom Hexamer Primer, 600 pmol/μl不超过4 kb25°C下10min，之后55°C下30min超过4 kb25°C下10min，之后50°C下60min⑦加热至85°C维持5min使Transcriptor Reverse Transcriptase失活。
∙将反应管置于冰上以终止反应。
∙反应管在+2~+8°C下可保存1-2小时，在-15~-25°C下可保存更长时间。
∙⑧此cDNA产物无需经过纯化即可用于PCR反应。
一般来说，使用1-5 μl 第一链cDNA作为模板加入PCR体系中。
若是最初的实验，可尝试在50 μl PCR体系中使用2 μl cDNA作为模板。
∙如果在LightCycler System上进行PCR，在20 μl 的反应体系中使用2-5 μl cDNA或cDNA稀释液。