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.
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
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 — 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 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.
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.
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.
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.
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.
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").
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
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)