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Sensiscript RT Kit (200)

Cat. No. / ID:  205213

For 200 reverse-transcription reactions: Sensiscript 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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Sensiscript 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。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 灵敏检测低至10个拷贝的模板
  • 通过高亲和力酶获得高产量cDNA
  • 无需RNase H消化步骤
  • 快速、简单的操作流程,无需繁琐的移液步骤

Product Details

Sensiscript Reverse Transcriptase (RT) Kit非常适合使用极少量的RNA(少于50 ng)进行高灵敏度的应用,如单细胞RT-PCR和活组织及LMD样本分析。Sensiscript Reverse Transcriptase对RNA具有高亲和力,可在宽动态范围内进行高效RT-PCR,对少量RNA进行极灵敏的RT-PCR。Sensiscript RT Kit包含Sensiscript Reverse Transcriptase、dNTPs和优化的反应缓冲液。只需加入引物就可进行快速、简单的cDNA合成。

Performance

Sensiscript Reverse Transcriptase (RT)在宽动态范围内具有很高的效率,对少量RNA也具有高灵敏度(参见" RT-PCR with RNA corresponding to 1 cell)。GC含量高的RNA区域可能导致其他逆转录酶停止,从RNA模板上脱落或跳过RNA的环状区(参见" Full-length RT-PCR products — B")。然而,QIAGEN逆转录酶对这些难扩增的模板均可扩增(参见see figure " Full-length RT-PCR products — A")。Sensiscript RT Kit无需优化,逆转录过程几乎没有困难。

QIAGEN具有严格的质量控制,保证Sensiscript RT Kit批间的重复性。Sensiscript RT Kits包含的经优化的Buffer RT、dNTPs和水,都保证不含RNase,每个批次的Sensiscript RT都对RT-PCR的重复性进行了全面的检测。

See figures

Principle

Sensiscript Reverse Transcriptase (RT)是第一个用于高灵敏度应用的商业化酶,可处理极少量的RNA(<50 ng),如单细胞RT-PCR或少量活组织分析。即用型Sensiscript RT对RNA具有高亲和力,配合dNTPs和经优化的反应缓冲液,可对低拷贝转录本进行高特异性和灵敏度的RT-PCR,还能读通复杂的RNA二级结构,无需调整温度或反应条件。请注意,不提供引物混合物。

处理病毒RNA,可选用Omniscript RT,因为大部分病毒RNA样本都含有载体RNA。

Procedure

使用经优化的Sensiscript Reverse Transcriptase反应缓冲液,无需繁琐的移液和预孵育步骤,也无需RNase H消化步骤(参见" Sensiscript Reverse Transcriptase Procedure")。
See figures

Applications

Sensiscript Reverse Transcriptase适用于以下应用:

  • 标准RT和RT-PCR
  • 定量real-time RT-PCR
  • 差异显示RT-PCR
  • 引物延伸和RACE分析
  • 合成用于克隆的双链cDNA

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationscDNA synthesis, RT-PCR, qRT-PCR
Reaction typeReverse transcription
Enzyme activityReverse transcription
Real-time or endpointEndpoint
MastermixNo
Sample/target typeRNA template
Single or multiplexSingle
With/without hotstartWithout hotstart

Resources

用户开发的实验方案 (1)
The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A+ mRNA.
试剂盒操作手册 (1)
Sensiscript Reverse Transcriptase for First-strand cDNA synthesis using <50 ng RNA; Two-tube RT-PCR; One-tube RT-PCR
安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
快速启动实验方案 (1)
Certificates of Analysis (1)
Kit Handbooks (1)
Sensiscript Reverse Transcriptase for First-strand cDNA synthesis using <50 ng RNA; Two-tube RT-PCR; One-tube RT-PCR
Quick-Start Protocols (1)
User-Developed Protocols (1)
The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A+ mRNA.

FAQ

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
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