QIAGEN Reverse Transcriptases

QIAGEN Omniscript and Sensiscript Reverse Transcriptases are unique recombinant enzymes with a high affinity for RNA, providing highly specific and sensitive reverse transcription without optimization.

Features and benefits

• Novel enzymes and unique buffers for high specificity
• Detect as few as 10 copies or start with as little as 1 pg RNA
• High yields of cDNA
• Robust systems guarantee consistent results

Principle

QIAGEN offers two recombinant reverse transcriptases with high affinity for RNA. This high affinity results in highly specific and sensitive RT-PCR even with low-copy transcripts, giving them the ability to read through even complex RNA secondary structures without adjusting temperature or reaction conditions (see figure "Comparison of Various Reverse Transcriptases").

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).

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"). These difficult templates, however, prove no problem for QIAGEN reverse transcriptases (see figure "Full-Length RT-PCR Products"). With no need for optimization, Omniscript and Sensiscript RT Kits make reverse transcription trouble-free.

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

Full-Length RT-PCR Products

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.  A  RT was carried out using the standard Omniscript RT protocol at the standard (37°C) and higher reaction temperatures as indicated.  B  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. (Data from reference 1)

Applications

QIAGEN Reverse Transcriptases are the ideal choice for gene-expression analysis and other applications such as:

• cDNA synthesis
• RT-PCR
• Quantitative RT-PCR
• Primer-extension and RACE analysis
• Differential display

Omniscript RT Kit

For standard reverse transcription

Component	Included
Omniscript Reverse Transcriptase
Buffer RT
dNTP Mix
RNase-free water

Omniscript Reverse Transcriptase is specially designed for all reverse transcription with any amount of RNA from 50 ng to 2 µg per reaction. A higher affinity for RNA results in higher sensitivity and superior performance even with low copy numbers (see figure "Sensitive RT-PCR of ≥10 Copies"). 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 (see figures"Comparison of Various Reverse Transcriptases" and "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.

操作手册:Omniscript Reverse Transcriptase Handbook (PDF version, 187 KB)