Omniscript RT Kit

反応あたり50 ng ～ 2 μg のRNA を用いた逆転写反応（エンドポイントPCR用）

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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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Reactions

200

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Omniscript 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。

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特徴

アフィニティーが高い酵素による高収量cDNA
わずか10コピーのテンプレートも高感度で検出
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製法

製品詳細

Omniscript Reverse Transcription（RT）Kitは、各反応あたり50 ngから2 µgのRNA量を用いた逆転写用に特別にデザインされています。RNAへのアフィニティーが高いOmniscript Reverse Transcriptaseは、他の逆転写酵素と比較して非常に優れた性能を示し、低いコピー数のテンプレートでさえ高感度なRT-PCRを実現します。Omniscript RT KitにはOmniscript 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

Omniscript Reverse Transcriptase （RT）の高いRNAアフィニティーによりコピー数の少ない転写物からでさえも、高効率および高感度のRT-PCR結果が得られるので（図" 10コピー以上の高感度なRT-PCR"）、温度あるいは反応条件の調整を行なわずに、複雑な二次構造を持つRNAでも、完全に読み取り、逆転写を実現します（図" 種々の逆転写酵素との比較"）。

その他の逆転写酵素では、GC含有率の高いRNA領域は逆転写が止まったりRNAテンプレートから解離したりする原因であり、またRNAのループ構造部分をスキップしてしまいます（図" 完全長の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、逆転写が効率よく行なわれることが証明されています（図" 完全長のRT-PCR産物 — A"）。Omniscript RT Kitは、至適化なしで事実上問題なく逆転写が可能なキットです。

図参照

Sensitive RT-PCR of ≥10 copies.

Comparison of various reverse transcriptases.

Full-length RT-PCR products — B.

Full-length RT-PCR products — A.

原理

RNAに対して高いアフィニティーを有するOmniscript Reverse Transcriptase（RT）は、どのようなテンプレートでも効率的で高感度の逆転写が可能であり、高収量のcDNAが得られます。dNTPsと至適化済みの反応バッファーが同梱されているため即使用可能であり、RNAに対するOmniscript RTの高いアフィニティーと相まって、高GC含量または複雑な二次構造のテンプレートの読み取りが実現しています。プライマーミックスは同梱されていなのでご注意ください。

Omniscript RTは、各反応あたり50 ngから2 µg のRNA量を用いた、あらゆる逆転写用に特別にデザインされています。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は通常ウイルスRNA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。

Omniscript 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Omniscript RT Kitsに添付されている至適化済みのBuffer RT、dNTPs、水はRNaseフリーの保証付きで、Omniscript RTの各ロットは、RT-PCRの再現性に関する多数の品質管理テストを受けています。

操作手順

Omniscript Reverse Transcriptaseの至適化済みの反応バッファーによって、手間のかかるピペッティングやプレインキュベーションステップが不要となり、RNase Hによる追加分解ステップも必要ありません（フローチャート""）。

図参照

Omniscript Reverse Transcriptase procedure.

アプリケーション

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

エンドポイントPCR用cDNA合成
クローニング用二本鎖cDNA合成
RACE (Rapid Amplification of cDNA Ends)
リニアRNA増幅
RNAの指数的増幅
SAGE (Serial Analysis of Gene Expression)
マイクロアレイ用標識反応
プライマーエクステンションによる転写開始部位解析

裏付けデータと数値

Full-length RT-PCR products — A.

RT-PCR was carried out with total RNA from maize leaves using the indicated RT reaction temperatures. A 1.2 kb fragment of the GC-rich maize gl2 transcript was amplified. RT was carried out using the standard Omniscript RT protocol at the standard (37°C) and higher reaction temperatures as indicated.

Specifications

Features	Specifications
ApplicationsJA	RT-PCR, qRT-PCR, primer-extension, RACE analysis
Real-time or endpoint	Endpoint
Mastermix	No
Enzyme activity	Reverse transcription
Single or multiplex	Single
With/without hotstart	Without hotstart
Reaction type	Reverse transcription
Sample/target type	RNA template

リソース

Download Safety Data Sheets for QIAGEN product components.

Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR

FAQ

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

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

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

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