Sensiscript RT Kit

For reverse transcription of less than 50 ng RNA per reaction for end-point PCR

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Sensiscript RT Kit (200)

Cat. No. / ID: 205213

For 200 reverse-transcription reactions: Sensiscript 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

Reactions

200

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The Sensiscript 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

Features

Sensitive detection of as few as 10 copies of template
High cDNA yields due to high-affinity enzyme
No additional RNase H digestion step required
Fast and easy procedure with no tedious pipetting steps

Product Details

The Sensiscript Reverse Transcriptase (RT) Kit is ideally suited for highly sensitive applications using very small amounts of RNA (less than 50 ng), such as single-cell RT-PCR and analysis of biopsies and LMD samples. Sensiscript Reverse Transcriptase allows highly efficient RT-PCR over a wide dynamic range and extremely sensitive RT-PCR with very small RNA amounts due to its high affinity for RNA. The Sensiscript RT Kit includes Sensiscript Reverse Transcriptase, dNTPs, and an optimized reaction buffer. Simply add primers for fast and easy cDNA synthesis.

Performance

Sensiscript Reverse Transcriptase (RT) 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"). 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 Sensiscript RT Kit makes reverse transcription virtually trouble-free.

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

See figures

RT-PCR with RNA corresponding to 1 cell.

Full-Length RT-PCR products — B.

Full-Length RT-PCR products — A.

Principle

Sensiscript Reverse Transcriptase (RT) 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. It is provided ready-to-use with dNTPs and in an optimized reaction buffer that, together with the high affinity of Sensiscript RT for RNA, results in highly specific and sensitive RT-PCR, even with low-copy transcripts, and the ability to read through even complex RNA secondary structures without adjusting temperature or reaction conditions. Please note that the primer mix is not provided.

For viral RNA, Omniscript RT is usually the enzyme of choice due to the presence of carrier RNA in most viral RNA preparations.

Procedure

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

See figures

Sensiscript Reverse Transcriptase procedure.

Applications

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

Standard RT and RT-PCR
Quantitative, real-time RT-PCR
Differential display RT-PCR
Primer extension and RACE analysis
Synthesis of double-stranded cDNA for cloning

Supporting data and figures

Full-Length RT-PCR products — A.

Specifications

Features	Specifications
Applications	cDNA synthesis, RT-PCR, qRT-PCR
Reaction type	Reverse transcription
Enzyme activity	Reverse transcription
Real-time or endpoint	Endpoint
Mastermix	No
Sample/target type	RNA template
Single or multiplex	Single
With/without hotstart	Without hotstart

Resources

The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A⁺ mRNA.

Sensiscript Reverse Transcriptase for First-strand cDNA synthesis using <50 ng RNA; Two-tube RT-PCR; One-tube RT-PCR

FAQ

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

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

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

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

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

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

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

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

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

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