Sensiscript RT Kit

反応あたり50 ng 未満のRNA を用いた逆転写反応

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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

Inquire

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。

✓ オンライン注文による24時間年中無休の自動処理システム

✓ 知識豊富で専門的な製品&テクニカルサポート

✓ 迅速で信頼性の高い（再）注文

特徴

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

製品詳細

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。

パフォーマンス

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。

図参照

RT-PCR with RNA corresponding to 1 cell.

Full-Length RT-PCR products — B.

Full-Length RT-PCR products — A.

原理

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。

操作手順

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""参照）。

図参照

Sensiscript Reverse Transcriptase procedure.

アプリケーション

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

裏付けデータと数値

Full-Length RT-PCR products — A.

Specifications

Features	Specifications
Applications	cDNA synthesis, RT-PCR, qRT-PCR
Reaction type	Reverse transcription
Enzyme activity	Reverse transcription
Real-time or endpoint	Endpoint
Mastermix	No
Sample/target type	RNA template
Single or multiplex	Single
With/without hotstart	Without hotstart

リソース

The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A⁺ mRNA.

Sensiscript Reverse Transcriptase for First-strand cDNA synthesis using <50 ng RNA; Two-tube RT-PCR; One-tube RT-PCR

Download Safety Data Sheets for QIAGEN product components.

FAQ

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

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

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

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

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

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

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

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

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

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