Omniscript RT Kit

For reverse transcription of 50 ng to 2 μg RNA per reaction for end-point PCR

  • High cDNA yields due to high-affinity enzyme
  • Sensitive detection of as few as 10 copies of template
  • No additional RNase H digestion step required
  • Fast and easy procedure with no tedious pipetting steps
The Omniscript Reverse Transcription (RT) Kit is specially designed for reverse transcription with any amount of RNA from 50 ng to 2 μg per reaction. A high affinity for RNA allows Omniscript Reverse Transcriptase to provide superior performance compared with other reverse transcriptases, delivering higher sensitivity in RT-PCR, even with low-copy numbers. The Omniscript RT Kit includes Omniscript Reverse Transcriptase, dNTPs and an optimized reaction buffer. Simply add primers for fast and easy cDNA synthesis.
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製品名 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
¥35,000
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
¥110,000

Omniscript RT Kit  は分子生物学実験用です。疾病の診断、治療または予防の目的には使用することはできません。


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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.|
Desempenho

The high affinity of Omniscript Reverse Transcriptase (RT) results in highly specific and sensitive RT-PCR, even with low-copy transcripts (see figure "Sensitive RT-PCR of ≥10 copies"), and the ability to read through even complex RNA secondary structures without adjusting temperature or reaction conditions (see figure "Comparison of various reverse transcriptases").

Regions of RNA with high GC content can cause other reverse transcriptases to stop, dissociate from the RNA template, or skip over looped-out regions of RNA (see figure "Full-length RT-PCR products — B"). These difficult templates, however, prove no problem for QIAGEN reverse transcriptases (see figure "Full-length RT-PCR products — A"). With no need for optimization, the Omniscript RT Kit makes reverse transcription virtually trouble-free.

Fundamento

Omniscript Reverse Transcriptase (RT) has a high affinity for RNA, which enables efficient and sensitive reverse transcription of any template, leading to high yields of cDNA. It is provided ready-to-use with dNTPs and in an optimized reaction buffer that, together with the high affinity of Omniscript RT for RNA, enables read-through of templates with high GC content or complex secondary structure. Please note that the primer mix is not provided.

Omniscript RT is specially designed for all reverse transcription with any amount of RNA from 50 ng to 2 µg per reaction. Omniscript RT is also usually the enzyme of choice with viral RNA due to the presence of carrier RNA in most viral RNA preparations. In comparative experiments, Omniscript RT consistently outperforms other reverse transcriptases over a wide range of starting RNA amounts.

Lot-to-lot reproducibility of Omniscript Kits is ensured by rigorous quality control at QIAGEN. The optimized Buffer RT, dNTPs, and water included in all Omniscript RT Kits are guaranteed RNase-free, and each lot of Omniscript RT is thoroughly tested for RT-PCR reproducibility.

Procedimento
With the optimized Omniscript Reverse Transcriptase reaction buffer, tedious pipetting and pre-incubation steps are eliminated, and no additional RNase H digestion step is needed (see flowchart "Omniscript Reverse Transcriptase Procedure").
Aplicações

Omniscript Reverse Transcriptase is suitable for use in the following applications:

  • cDNA synthesis for end-point PCR
  • Synthesis of double-stranded cDNA for cloning
  • RACE (Rapid Amplification of cDNA Ends)
  • Linear RNA amplification
  • Exponential RNA amplification
  • SAGE (Serial Analysis of Gene Expression)
  • Labeling for microarrays
  • Analysis of transcription start site by primer extension
Feature
Specifications
Applications RT-PCR, qRT-PCR, primer-extension, RACE analysis
Enzyme activity Reverse transcription
Mastermix No
Reaction type Reverse transcription
Real-time or endpoint Endpoint
Sample/target type RNA template
Single or multiplex Single
With/without hotstart Without hotstart

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Kit Handbooks
1
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR
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画像
Full-Length RT-PCR Products.
完全長のRT-PCR産物 — A
表記の逆転写反応の温度でトウモロコシの葉から得られたトータルRNAを用いてRT-PCRを行なった。GC含有率の高いトウモロコシgl2転写物の1.2 kbフラグメントを増幅した。スタンダードOmniscript RTプロトコールを用いて、標準温度(37℃)および表記より高い反応温度で逆転写を行なった。
Full-Length RT-PCR Products.
完全長のRT-PCR産物 — B
表記の逆転写反応の温度でトウモロコシの葉から得られたトータルRNAを用いてRT-PCRを行なった。GC含有率の高いトウモロコシgl2転写物の1.2 kbフラグメントを増幅した。スタンダードMMLV RNase H- RTプロトコール(Supplier L)を用いて標準温度(42℃)および表記の反応温度でRTを行なった。このスタンダードプロトコールでは、まず必要な主要変性ステップ、そして逆転写後推奨されているRNase H分解ステップを行なった。
Omniscript RT procedure
Omniscript Reverse Transcriptase製法

Omniscript RT Kitには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 Reverse Transcriptase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る。この事前の至適化により、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による追加の分解ステップも必要ない。

Superior sensitivity and dynamic range of Omniscript RT
種々の逆転写酵素との比較
各社のプロトコールに従い、種々の逆転写酵素を用いて逆転写を行なった。反応に利用したHeLa細胞のトータルRNA量を図の上部に示した。QIAGEN Taq DNAポリメラーゼを用いて逆転写反応液の1/20量からPCR増幅を25サイクル行なった。1.7 kbのβ-Actinフラグメントを増幅した。 Omniscript::Omniscript Reverse Transcriptase(QIAGEN)、 MMLV RNase H–:モロニーマウス白血病ウイルス由来RNase H– reverse transcriptase(Supplier L)、 MMLV:野生型MMLV reverse transcriptase(Supplier L)、 AMV RNase H–:トリ骨髄芽球症ウイルス由来熱安定性RNase H– reverse transcriptase(Supplier L)、 AMV:野生型AMV reverse transcriptase(Supplier P)。
Sensitive RT-PCR of ≥10 Copies
10コピー以上の高感度なRT-PCR
10~104コピーに相当する700 ntのin vitro転写物を含むテンプレート(トータルRNA量200 ng)を逆転写後、PCRを行なった。各逆転写反応液の1/4(2.5から2500 cDNAコピーに相当)を330 bpフラグメントの増幅に用いた。