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)

  • 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
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。
Product Cat. no. List price:
Omniscript RT Kit (50)
For 50 reverse-transcription reactions: 200 units Omniscript Reverse Transcriptase, 150 µl 10x Buffer RT, 100 µl dNTP Mix (contains 5 mM each dNTP), 1.1 ml RNase-free water
205111
260,00 €
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Omniscript RT Kit (200)
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
205113
885,00 €
Add to cart
The Omniscript RT Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Full-length RT-PCR products — A.|Full-length RT-PCR products — B.|Omniscript Reverse Transcriptase procedure.|Comparison of various reverse transcriptases.|Sensitive RT-PCR of ≥10 copies.|
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.
|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 MMLV RNase H- RT protocol (Supplier L) at the standard (42°C) and other reaction temperatures, as indicated. This standard protocol was carried out with the required preliminary denaturation step and the recommended RNase H digest step following RT.
|The Omniscript RT Kit includes dNTPs and an optimized reaction buffer that, together with the high affinity of Omniscript Reverse Transcriptase for RNA, enables read-through of templates with high GC content or complex secondary structures. Due to this pre-optimization, tedious pipetting and pre-incubation steps are eliminated, and no additional RNase H digestion step is required.|Reverse transcription was carried out with different reverse transcriptases according to suppliers' specifications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplification with QIAGEN Taq DNA Polymerase. A 1.7 kb beta-actin fragment was amplified. Omniscript: Omniscript Reverse Transcriptase (QIAGEN); MMLV RNase H–: RNase H– reverse transcriptase from Moloney murine leukemia virus (Supplier L); MMLV: wild-type MMLV reverse transcriptase (Supplier L); AMV RNase H–: thermostable RNase H– reverse transcriptase from avian myeloblastosis virus (Supplier L); AMV: wild-type AMV reverse transcriptase (Supplier P).|A 700 nt in vitro transcript was reverse transcribed from 10 to 104 copies of starting template (in 200 ng total RNA), followed by PCR. 1/4 of each reverse-transcription reaction (corresponding to 2.5 to 2500 cDNA copies) was used for PCR amplification of a 330 bp fragment.|
Performance

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。

Principle

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。

Procedure
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"Omniscript Reverse Transcriptase製法")。
Applications

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

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Kit Handbooks
1
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR
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Safety Data Sheets
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Full-Length RT-PCR Products.
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.
Full-Length RT-PCR Products.
Full-length RT-PCR products — B.
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 MMLV RNase H- RT protocol (Supplier L) at the standard (42°C) and other reaction temperatures, as indicated. This standard protocol was carried out with the required preliminary denaturation step and the recommended RNase H digest step following RT.
Omniscript RT procedure
Omniscript Reverse Transcriptase procedure.

The Omniscript RT Kit includes dNTPs and an optimized reaction buffer that, together with the high affinity of Omniscript Reverse Transcriptase for RNA, enables read-through of templates with high GC content or complex secondary structures. Due to this pre-optimization, tedious pipetting and pre-incubation steps are eliminated, and no additional RNase H digestion step is required.

Superior sensitivity and dynamic range of Omniscript RT
Comparison of various reverse transcriptases.
Reverse transcription was carried out with different reverse transcriptases according to suppliers' specifications, using the indicated amounts of total RNA from HeLa cells. 1/20 of the reverse-transcription reaction was used in a 25-cycle PCR amplification with QIAGEN Taq DNA Polymerase. A 1.7 kb beta-actin fragment was amplified. Omniscript: Omniscript Reverse Transcriptase (QIAGEN); MMLV RNase H–: RNase H– reverse transcriptase from Moloney murine leukemia virus (Supplier L); MMLV: wild-type MMLV reverse transcriptase (Supplier L); AMV RNase H–: thermostable RNase H– reverse transcriptase from avian myeloblastosis virus (Supplier L); AMV: wild-type AMV reverse transcriptase (Supplier P).
Sensitive RT-PCR of ≥10 Copies
Sensitive RT-PCR of ≥10 copies.
A 700 nt in vitro transcript was reverse transcribed from 10 to 104 copies of starting template (in 200 ng total RNA), followed by PCR. 1/4 of each reverse-transcription reaction (corresponding to 2.5 to 2500 cDNA copies) was used for PCR amplification of a 330 bp fragment.