Omniscript RT Kit

在终点法PCR中,用于RNA模板量为50 ng到2 μg的逆转录反应

S_1225_GEF_PCR0019
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Omniscript RT Kit (200)

Cat. No. / ID:  205113

For 200 reverse-transcription reactions: 800 units Omniscript Reverse Transcriptase, 4 x 150 µl 10x Buffer RT, 4 x 100 µl dNTP Mix (contains 5 mM each dNTP), 4 x 1.1 ml RNase-free water
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Omniscript RT Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM

Features

  • 高亲和力酶获得高产量cDNA
  • 灵敏的检测低至10拷贝的模板
  • 无需额外的RNase H消化
  • 快速简单的操作流程,无需繁琐的移液步骤

Product Details

Omniscript Reverse Transcription (RT) Kit专为单次逆转录反应中使用50 ng至2 μg RNA模板而设计。与RNA的高亲和力使得Omniscript Reverse Transcriptase比其它逆转录酶表现更出色,即使是低拷贝模板,在RT-PCR中也能实现更高的灵敏度。Omniscript RT Kit包括Omniscript Reverse Transcriptase、dNTPs和优化的反应缓冲液。只需加入引物,即可简单快速合成cDNA。

Performance

高亲和力Omniscript Reverse Transcriptase (RT)在RT-PCR中具有高特异性和高灵敏度,即使是低拷贝转录(参见" Sensitive RT-PCR of ≥10 copies"),无需调整温度或反应条件即可通读复杂的RNA二级结构(参见" Comparison of various reverse transcriptases")。

GC含量高的RNA区域可能导致逆转录反应停止、从RNA模板脱离或跳过RNA环外区域(参见" Full-length RT-PCR products — B")。经证明,这些难扩增模板对QIAGEN逆转录酶没有影响(参见"F ull-length RT-PCR products — A")。无需优化,Omniscript RT Kit就可使逆转录反应顺利进行。

See figures

Principle

Omniscript Reverse Transcriptase (RT)与RNA的高亲和力确保多种模板逆转录反应的高效性和灵敏性,获得高产量cDNA。提供即用型dNTPs,经优化的反应缓冲液和与RNA高亲和力的Omniscript RT,可通读GC含量高或复杂二级结构的模板。请注意,不提供引物混合物。

Omniscript RT专为逆转录反应设计,单次反应使用50 ng到2 µg的RNA模板。另可作为病毒RNA的酶选择,因为在多数病毒RNA制备中存在载体RNA。在对比实验中,Omniscript RT的表现在一系列不同RNA起始量中都优于其它逆转录酶。

QIAGEN提供严格的质量控制,确保Omniscript Kits批与批之间的高重复性。所有Omniscript RT Kits都包括经优化的Buffer RT、dNTPs和纯水,保证不含RNase、每批Omniscript RT都进行全面得RT-PCR重复性测试。

Procedure

使用经优化的Omniscript Reverse Transcriptase反应缓冲液,无需繁琐的移液和预培养步骤,无需另外使用RNase H消化步骤(参见" Omniscript Reverse Transcriptase Procedure")。
See figures

Applications

Omniscript Reverse Transcriptase适合以下应用:

  • 合成cDNA ,用于终点式PCR
  • 合成双链cDNA,用于克隆
  • RACE(cDNA终点的快速扩增)
  • 线性RNA扩增
  • 指数型RNA扩增
  • SAGE(基因表达序列分析)
  • 微阵列标记
  • 引物延伸转录起始位点分析

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsRT-PCR, qRT-PCR, primer-extension, RACE analysis
Real-time or endpointEndpoint
MastermixNo
Enzyme activityReverse transcription
Single or multiplexSingle
With/without hotstartWithout hotstart
Reaction typeReverse transcription
Sample/target typeRNA template

Resources

安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
试剂盒操作手册 (1)
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR
Certificates of Analysis (1)
Kit Handbooks (1)
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR

FAQ

How should I store cDNA produced using Omniscript Reverse Transcriptase?
We recommend to store the cDNA at -20°C in aliquots to avoid repeated freeze/thaw cyles. If the cDNA requires dilution, we recommend to dilute it in Tris buffer, pH 8.0. Avoid storing the aliquots at high dilutions. If possible, use siliconized tubes for storage of cDNA dilutions to avoid adsorption of nucleic acids to the tube walls.
FAQ ID -561
Are there specific recommendations for performing RT-PCR on RNA isolated from paraffin-embedded samples?

Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure. 

If performing RT-PCR with degraded RNA, we recommend using gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation.

We recommend that the RNeasy FFPE or miRNeasy FFPE kit be used to isolate the RNA.

FAQ ID -828
Do I need to use an RNase inhibitor in my RT reaction?
To be on the safe side, we highly recommend the use of RNase inhibitors in all RT reactions, since RNases are nearly everywhere and it is very easy to contaminate samples during reaction setup. The reaction conditions used for RT are well-suited for RNase activity. Even traces of RNase can nick the RNA, causing shortened cDNA products, low yields, and reduced RT-PCR sensitivity.
FAQ ID -119
Do you recommend 1-step or 2-step real-time RT-PCR for gene expression analysis?

In one-step RT-PCR, both reverse transcription and amplification are performed in the same tube. Upon completion of reverse transcription, the reaction temperature is raised to reach denaturation/PCR enzyme activation temperature and the thermal cycling (PCR) begins. One-step RT-PCR generally uses gene-specific primers for both the RT and PCR steps. The procedure is fast, easy to automate, and minimizes the risk of contamination due to fewer handling steps.

In two-step RT-PCR, the RNA is first transcribed into cDNA using oligo-dT primers, random oligos, or gene-specific primers. An aliquot of the RT reaction is subsequently added to the real-time PCR reaction in a second tube. Choice of different types of RT primers allows the analysis of different transcripts by PCR from one RT reaction. Most commonly, an oligo-dT primer is used for the RT step, followed by PCR with a pair of gene-specific primers. Precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage.

 

The advantages of each method are summarized below:

Two-step RT-PCR One-step RT-PCR
Multiple PCRs from one RT reaction Easy handling
Flexibility with RT primer choice Fast procedure
Enables long-term storage of cDNA High reproducibility
  Low contamination risk

 

Optimized one-step and two-step RT-PCR kits compatible with any real-time cycler are available from QIAGEN. For further details, please see our online section on 'Critical factors for successful gene expression assays', or download our Brochure 'Critical Factors for Successful Real-Time PCR'.

FAQ ID -1056
Is mRNA isolation necessary for sensitive RT-PCR?
Usually not. Since RT-PCR is extremely sensitive, as little as 10–200 ng total RNA is sufficient for each 25–50 µl reaction mix, depending on the RT system. For abundant mRNA species, it is possible to use even less than 10 ng total RNA. For rare mRNA species, use a sequence-specific primer in the RT reaction to increase sensitivity. RNA content in various cells and tissues can be found here.
FAQ ID -111
Do you have a protocol for radioactive labelling of cDNA?

Yes, please follow the User-Developed Protocol 'Labelling of cDNA using labeled [alpha-32P] dCTP and 1 µg RNA with the Omniscript RT Kit' (RT08). 

 

Please contact QIAGEN Technical Service for this protocol.

FAQ ID -960
Why is the RT step with the QuantiFast RT Kits much shorter compared to QuantiTect RT Kits?

The combination of Omniscript and Sensiscript Reverse Transcriptases was optimized in the QuantiFast RT-PCR Kits. In addition, an optimized dNTP concentration and the limitation of amplicon size to <300 bp allow to reduce the time for the reverse transcription step to only 10 minutes.

 

FAQ ID -1451
What is the recommended solution in which to store RNA samples that will be used as templates for cDNA synthesis?
For best results, all RNA samples should be suspended in RNase-free water. Alternatively, RNase-free 1 mM sodium citrate (pH 6.5) or 10 mM Tris buffer (pH 7.0) may be used. Do not use DEPC-treated water, as most DEPC preparations are contaminated with molecules that are inhibitory to reverse transcription and/or PCR. For long-term storage, RNA preps may be stored at -70 ºC in RNase-free water, or the buffers listed above, or precipitated in ethanol or isopropanol. In order to avoid repeated freeze-thaw cycles, it is recommended that frozen RNA samples be stored as multiple, single-use aliquots.
FAQ ID -2659
Can I use Omniscript or Sensiscript RT's at a higher temperature?
Omniscript and Sensiscript RTs are fully active at 37°C. These enzymes have high affinity for RNA, allowing reverse transcription without the need for higher temperatures. Therefore, for optimal results, we recommend carrying out all RT reactions with Omniscript or Sensiscript at 37°C. Only in rare cases, where further optimization is needed, may it be effective to raise the temperature to 42°C or 50°C although a slight reduction in RT activity and half-life may occur at these temperatures.
FAQ ID -116
Should I use Omniscript or Sensiscript for reverse transcription of low-copy mRNA?
Omniscript and Sensiscript RT are recombinant RTs optimized for use with different amounts of starting RNA. Both are sensitive for detecting low-copy mRNA species. The enzyme of choice depends on the total amount of RNA in the sample (including any carrier RNA present), regardless of the specific target RNA copy number. For standard reverse transcription, with 50 ng to 2 µg of RNA per reaction, Omniscript RT provides optimal results for both high- and low-copy mRNA species. Sensiscript RT is optimized for use with very small amounts of RNA (<50 ng), such as for single-cell RT-PCR or analysis of small biopsies.
FAQ ID -297
Do you have a protocol for Cyanine 570, Cyanine 670, or biotin labeling of cDNA?

Yes, please follow either of the User-Developed Protocols:

  • 'Labelling of cDNA using labeled dUTP and <50 ng RNA with the Sensiscript RT Kit' (RT05)
  • 'Labelling of cDNA using labeled dUTP and 50 ng-2 µg RNA with the Omniscript RT Kit' (RT06)
  • 'Labelling of cDNA using labeled dUTP and 5-50 µg RNA with the Omniscript RT Kit' (RT07)

 

  • 'Labeling of cDNA using labeled dCTP and <50 ng RNA with the Sensiscript RT Kit' (RT02)
  • 'Labeling of cDNA using labeled dCTP and 50 ng-2 µg RNA with the Omniscript RT Kit' (RT03)
  • 'Labeling of cDNA using labeled dCTP and 5-50 µg RNA with the Omniscript RT Kit' (RT04)

Please contact QIAGEN Technical Service for these protocols.

FAQ ID -959
A white precipitate has formed in my 10x RT buffer. Is it still ok to use?
Yes. Precipitates may form when the buffer freezes. We recommend that you thaw the buffer on ice, then vortex the tube at room temperature until the precipitate has re-dissolved. Do not centrifuge the tube. Do not heat the buffer.
FAQ ID -216