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

Sensiscript RT Kit

For reverse transcription using small amounts of RNA (<50 ng RNA including carrier RNA)

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

Sensiscript Reverse Transcriptase is the first commercially available enzyme designed for highly sensitive applications with very small amounts of RNA (<50 ng), such as single-cell RT-PCR or analysis of small biopsies. Sensiscript Reverse Transcriptase provides outstanding efficiency over a wide dynamic range and high sensitivity for very small amounts of RNA (see figure "RT-PCR with RNA Corresponding to 1 Cell"). For viral RNA, however, Omniscript RT is usually the enzyme of choice due to the presence of carrier RNA in most viral RNA preparations.

RT-PCR with RNA Corresponding to 1 Cell

Total RNA was isolated from 10 to 5000 HeLa cells using RNeasy technology. 1/10 of each eluate (corresponding to RNA from 1 to 500 cells) was used for reverse transcription with Sensiscript Reverse Transcriptase. 1/2 of each RT reaction was then used in a 40-cycle PCR with QIAGEN Taq DNA Polymerase and primers specific for genes encoding β-actin, NF-κB, inositol-1,4,5-triphosphate receptor, or dystrophin. Sizes of the amplicons are as indicated.

操作手册:Sensiscript Reverse Transcriptase Handbook (PDF version, 191 KB)

Ordering Information Product Contents Cat. No. Sensiscript RT Kit (50) For 50 reverse-transcription reactions: Sensiscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,* RNase-free water 205211 Sensiscript RT Kit (200) For 200 reverse-transcription reactions: Sensiscript Reverse Transcriptase, 10x Buffer RT, dNTP Mix,* RNase-free water 205213