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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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¥125,000
Reactions
200
50
Sensiscript RT Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • わずか10 コピーのテンプレートも高感度で検出
  • RNA への親和性が高い酵素で高収量 cDNA
  • 追加の RNase H 分解は不要
  • 煩わしいピペッティング操作なしの迅速で簡単な方法

Product Details

Sensiscript Reverse Transcriptase (RT) Kit は、シングルセルRT-PCR、バイオプシーやLMD サンプルの解析のような非常に微量なRNA(50 ng 未満)を用いた高感度アプリケーションに最適です。Sensiscript Reverse TranscriptaseはRNAに対する高い親和性を持つため、幅広いダイナミックレンジで効率的なRT-PCR を実現し、微量RNAでも非常に高感度なRT-PCR が可能です。Sensiscript RT Kitには、Sensiscript Reverse Transcriptase、dNTPs、至適化済みの反応バッファーが含まれています。プライマーを添加するだけで迅速かつ簡単にcDNAを合成できます。

Performance

Sensiscript Reverse Transcriptase(RT)は、幅広いダイナミックレンジにわたる高い効率と、微量のRNAに対する高い感度をもたらします(図" 1個の細胞に相当するRNAを用いたRT-PCR")。その他の逆転写酵素では、GC含有率の高いRNA領域は逆転写酵素が止まったり、RNAテンプレートから解離する原因であり、またRNAのループ構造部分をスキップしてしまいます(図 " 完全な長さのRT-PCR産物 - B")。しかし、このような逆転写が困難なテンプレートも、QIAGENの逆転写酵素を使用すれば、逆転写反応が効率よく行なわれることが証明されています(図 " 完全な長さのRT-PCR産物 - A")。Sensiscript RT Kit は、至適化を必要とせず、ほとんど問題なく逆転写反応を行なえるキットです。

Sensiscript RT Kitsのロット間の再現性は、QIAGENの厳しい品質管理により保証されています。全てのSensiscript RT Kitsに添付されている最適化されたBuffer RT、dNTP、および水は、RNaseフリーを保証されています。Sensiscript RTの各ロットは、RT-PCRの再現性についての品質管理テストを徹底的に受けています。

See figures

Principle

Sensiscript Reverse Transcriptase(RT)は、シングルセルRT-PCRやバイオプシーサンプル解析など、微量のRNA(50 ng以下)を使用する高感度アプリケーションのためにデザインされ、市販された最初の酵素です。RNAに対するSensiscript RTの高い親和性とともに、低コピー転写物を使用しても高特異性かつ高感度のRT-PCRと、温度または反応条件の調節がなくても、複雑な二次構造をもつRNAでも、完全に読み取りを実現する至適化済みの反応バッファーで、dNTPと一緒にすぐに使用することができます。プライマーミックスが含まれていない点にご注意ください。

多くのウイルスRNA調製にはキャリアRNAが存在するので、Omniscript RTがウイルスRNAの逆転写反応に最適な酵素です。比較実験においても、Omniscript RTは、幅広いRNAのスタート量で他の逆転写酵素より一定した優れた性能を示します。

Procedure

至適化済みのSensiscript Reverse Transcriptase 反応バッファーを使用すれば、手間のかかるピペット操作とプレインキュベーションステップがなくなり、さらに、RNase H 分解ステップを追加する必要もなくなります(フローチャート" Sensiscript Reverse Transcriptase操作手順"参照)。
See figures

Applications

Sensiscript Reverse Transcriptaseは、以下のようなアプリケーションに最適です。

  • 標準的なPCRおよびRT-PCR
  • リアルタイム定量RT-PCR
  • ディファレンシャルディスプレイRT-PCR
  • プライマーエクステンションおよび RACE 解析
  • クローニング用二本鎖cDNA合成

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

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.
キットハンドブック (2)
Sensiscript Reverse Transcriptase for First-strand cDNA synthesis using <50 ng RNA; Two-tube RT-PCR; One-tube RT-PCR
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
クイックスタートプロトコール (1)
Certificates of Analysis (1)
Kit Handbooks (2)
Sensiscript Reverse Transcriptase for First-strand cDNA synthesis using <50 ng RNA; Two-tube RT-PCR; One-tube RT-PCR
Quick-Start Protocols (1)

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