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Omniscript RT Kit

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

Features

  • 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
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Omniscript RT Kit (200)

Cat. No. / ID: 205113

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
$1,038.00
Reactions
200
50
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.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

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.

Performance

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.

See figures

Principle

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.

Procedure

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").
See figures

Applications

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

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsRT-PCR, qRT-PCR, primer-extension, RACE analysis
Real-time or endpointEndpoint
MastermixNo
Enzyme activityReverse transcription
Single or multiplexSingle
With/without hotstartWithout hotstart
Reaction typeReverse transcription
Sample/target typeRNA template

Resources

Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Kit Handbooks (1)
Omniscript Reverse Transcriptase - For First-strand cDNA synthesis Two-tube RT-PCR

FAQ

How should I store cDNA produced using Omniscript Reverse Transcriptase?
We recommend to store the cDNA at -20°C in aliquots to avoid repeated freeze/thaw cyles. If the cDNA requires dilution, we recommend to dilute it in Tris buffer, pH 8.0. Avoid storing the aliquots at high dilutions. If possible, use siliconized tubes for storage of cDNA dilutions to avoid adsorption of nucleic acids to the tube walls.
FAQ ID -561
Are there specific recommendations for performing RT-PCR on RNA isolated from paraffin-embedded samples?

Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure. 

If performing RT-PCR with degraded RNA, we recommend using gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation.

We recommend that the RNeasy FFPE or miRNeasy FFPE kit be used to isolate the RNA.

FAQ ID -828
Do I need to use an RNase inhibitor in my RT reaction?
To be on the safe side, we highly recommend the use of RNase inhibitors in all RT reactions, since RNases are nearly everywhere and it is very easy to contaminate samples during reaction setup. The reaction conditions used for RT are well-suited for RNase activity. Even traces of RNase can nick the RNA, causing shortened cDNA products, low yields, and reduced RT-PCR sensitivity.
FAQ ID -119
Do you recommend 1-step or 2-step real-time RT-PCR for gene expression analysis?

In one-step RT-PCR, both reverse transcription and amplification are performed in the same tube. Upon completion of reverse transcription, the reaction temperature is raised to reach denaturation/PCR enzyme activation temperature and the thermal cycling (PCR) begins. One-step RT-PCR generally uses gene-specific primers for both the RT and PCR steps. The procedure is fast, easy to automate, and minimizes the risk of contamination due to fewer handling steps.

In two-step RT-PCR, the RNA is first transcribed into cDNA using oligo-dT primers, random oligos, or gene-specific primers. An aliquot of the RT reaction is subsequently added to the real-time PCR reaction in a second tube. Choice of different types of RT primers allows the analysis of different transcripts by PCR from one RT reaction. Most commonly, an oligo-dT primer is used for the RT step, followed by PCR with a pair of gene-specific primers. Precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage.

 

The advantages of each method are summarized below:

Two-step RT-PCR One-step RT-PCR
Multiple PCRs from one RT reaction Easy handling
Flexibility with RT primer choice Fast procedure
Enables long-term storage of cDNA High reproducibility
  Low contamination risk

 

Optimized one-step and two-step RT-PCR kits compatible with any real-time cycler are available from QIAGEN. For further details, please see our online section on 'Critical factors for successful gene expression assays', or download our Brochure 'Critical Factors for Successful Real-Time PCR'.

FAQ ID -1056
Is mRNA isolation necessary for sensitive RT-PCR?
Usually not. Since RT-PCR is extremely sensitive, as little as 10–200 ng total RNA is sufficient for each 25–50 µl reaction mix, depending on the RT system. For abundant mRNA species, it is possible to use even less than 10 ng total RNA. For rare mRNA species, use a sequence-specific primer in the RT reaction to increase sensitivity. RNA content in various cells and tissues can be found here.
FAQ ID -111
Do you have a protocol for radioactive labelling of cDNA?

Yes, please follow the User-Developed Protocol 'Labelling of cDNA using labeled [alpha-32P] dCTP and 1 µg RNA with the Omniscript RT Kit' (RT08). 

 

Please contact QIAGEN Technical Service for this protocol.

FAQ ID -960
Why is the RT step with the QuantiFast RT Kits much shorter compared to QuantiTect RT Kits?

The combination of Omniscript and Sensiscript Reverse Transcriptases was optimized in the QuantiFast RT-PCR Kits. In addition, an optimized dNTP concentration and the limitation of amplicon size to <300 bp allow to reduce the time for the reverse transcription step to only 10 minutes.

 

FAQ ID -1451
What is the recommended solution in which to store RNA samples that will be used as templates for cDNA synthesis?
For best results, all RNA samples should be suspended in RNase-free water. Alternatively, RNase-free 1 mM sodium citrate (pH 6.5) or 10 mM Tris buffer (pH 7.0) may be used. Do not use DEPC-treated water, as most DEPC preparations are contaminated with molecules that are inhibitory to reverse transcription and/or PCR. For long-term storage, RNA preps may be stored at -70 ºC in RNase-free water, or the buffers listed above, or precipitated in ethanol or isopropanol. In order to avoid repeated freeze-thaw cycles, it is recommended that frozen RNA samples be stored as multiple, single-use aliquots.
FAQ ID -2659
Can I use Omniscript or Sensiscript RT's at a higher temperature?
Omniscript and Sensiscript RTs are fully active at 37°C. These enzymes have high affinity for RNA, allowing reverse transcription without the need for higher temperatures. Therefore, for optimal results, we recommend carrying out all RT reactions with Omniscript or Sensiscript at 37°C. Only in rare cases, where further optimization is needed, may it be effective to raise the temperature to 42°C or 50°C although a slight reduction in RT activity and half-life may occur at these temperatures.
FAQ ID -116
Should I use Omniscript or Sensiscript for reverse transcription of low-copy mRNA?
Omniscript and Sensiscript RT are recombinant RTs optimized for use with different amounts of starting RNA. Both are sensitive for detecting low-copy mRNA species. The enzyme of choice depends on the total amount of RNA in the sample (including any carrier RNA present), regardless of the specific target RNA copy number. For standard reverse transcription, with 50 ng to 2 µg of RNA per reaction, Omniscript RT provides optimal results for both high- and low-copy mRNA species. Sensiscript RT is optimized for use with very small amounts of RNA (<50 ng), such as for single-cell RT-PCR or analysis of small biopsies.
FAQ ID -297
Do you have a protocol for Cyanine 570, Cyanine 670, or biotin labeling of cDNA?

Yes, please follow either of the User-Developed Protocols:

  • 'Labelling of cDNA using labeled dUTP and <50 ng RNA with the Sensiscript RT Kit' (RT05)
  • 'Labelling of cDNA using labeled dUTP and 50 ng-2 µg RNA with the Omniscript RT Kit' (RT06)
  • 'Labelling of cDNA using labeled dUTP and 5-50 µg RNA with the Omniscript RT Kit' (RT07)

 

  • 'Labeling of cDNA using labeled dCTP and <50 ng RNA with the Sensiscript RT Kit' (RT02)
  • 'Labeling of cDNA using labeled dCTP and 50 ng-2 µg RNA with the Omniscript RT Kit' (RT03)
  • 'Labeling of cDNA using labeled dCTP and 5-50 µg RNA with the Omniscript RT Kit' (RT04)

Please contact QIAGEN Technical Service for these protocols.

FAQ ID -959
A white precipitate has formed in my 10x RT buffer. Is it still ok to use?
Yes. Precipitates may form when the buffer freezes. We recommend that you thaw the buffer on ice, then vortex the tube at room temperature until the precipitate has re-dissolved. Do not centrifuge the tube. Do not heat the buffer.
FAQ ID -216