QIAGEN OneStep RT-PCR Kit

For highly sensitive and specific one-step RT-PCR

Products

QIAGEN OneStep RT-PCR Kit ist für molekularbiologische Anwendungen vorgesehen. Dieses Produkt ist nicht für die Diagnose, Prävention oder Behandlung einer Krankheit bestimmt.

QIAGEN OneStep RT-PCR Kit (100)

Cat. No. / ID: 210212

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)

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This product contains substances regulated under REACH (EC 1907/2006 Annex XIV). The use of this product in the EU is permitted subject to an exemption (Article 56(3)). Please refer to the REACH notification and the SDS of this product, both of which can be found in the “Resources” section of this page, for more information.

QIAGEN OneStep RT-PCR Kit (1000)

Cat. No. / ID: 210215

For 1000 x 50 µl reactions (available in a single tube): QIAGEN OneStep RT-PCR Enzyme Mix (2 x 1 ml), 5x QIAGEN OneStep RT-PCR Buffer (1 x 10 ml), dNTP Mix (2 x 1 ml, 10 mM each), 5x Q-Solution (1 x 10 ml), RNase-Free Water (1 x 40 ml)

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QIAGEN OneStep RT-PCR Kit (25)

Cat. No. / ID: 210210

For 25 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 50 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 250 µl), dNTP Mix (1 x 50 µl, 10 mM each), 5x Q-Solution (1 x 400 µl), RNase-Free Water (1 x 1.9 ml)

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Features

Fast and easy one-tube setup
Efficient one-step RT-PCR of any RNA template without optimization
Unique enzyme mix for high specificity and sensitivity
Balanced mixture of enzymes with optimized reverse-transcription buffer

Product Details

The QIAGEN One-Step RT-PCR Kit provides a blend of Sensiscript and Omniscript Reverse Transcriptases, HotStarTaq DNA Polymerase, QIAGEN OneStep RT-PCR Buffer, a dNTP mix, and Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates. The easy one-tube setup and optimized components result in highly sensitive and successful results.

Performance

The QIAGEN OneStep RT-PCR Kit provides a convenient format for highly sensitive and specific RT-PCR using any RNA template. The kit includes optimized components that allow both reverse transcription and PCR amplification to take place in the same reaction mix in a "one-step" reaction. A unique enzyme combination and specially developed reaction buffer ensure efficient, highly specific reverse transcription and PCR in one tube, without the need for optimization (see figures "" and ""). The innovative, dual-cation PCR buffer provided with the kit ensures high yields of specific PCR product over a wide range of annealing temperatures (see figure ""). Suboptimal RT-PCR is improved using Q-Solution, a unique additive that facilitates reverse transcription and amplification of templates with a high GC content or a high degree of secondary structure (see figure "").

See figures

Highly specific RT-PCR using low amounts of template.

Efficient detection of viral RNA.

Influence of annealing temperature on specificity.

RT-PCR of GC-rich template.

Principle

The QIAGEN OneStep RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template.

The QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend for both reverse transcription and PCR. The unique combination of Omniscript and Sensiscript Reverse Transcriptases, with their high affinity for RNA templates, ensures highly efficient and sensitive transcription of RNA amounts from as little as 1 pg up to 2 µg. After reverse transcription, reactions are heated to 95°C for 15 minutes to activate HotStarTaq DNA Polymerase and simultaneously inactivate the reverse transcriptases. This hot-start step eliminates nonspecific amplification products such as primer dimers and reduces background smear, ensuring highly sensitive and reproducible RT-PCR (see figures " Highly specific RT-PCR using low amounts of template" and " Efficient detection of viral RNA").

Wide range of annealing temperatures

The optimal primer annealing temperature is dependent on the base composition (i.e., the proportion of A, T, G, and C nucleotides), primer concentration, and ionic reaction environment. QIAGEN PCR Buffers contain both K⁺ and NH₄⁺ and deliver high yields of specific PCR product over a wide range of annealing temperatures (see figure " Increased specific primer annealing"). This specificity is achieved by destabilizing non-specifically bound primers, providing a more robust reaction environment and eliminating the need for tedious annealing temperature optimization. In contrast, the range of optimal PCR annealing temperatures is smaller and less predictable using a PCR or one-step RT-PCR buffer that only contains K⁺, as illustrated in figure " Influence of annealing temperature on specificity".

QIAGEN OneStep RT-PCR Buffer has been specially developed to allow both efficient reverse transcription and PCR amplification. The buffer contains novel additives that prevent inhibition of PCR amplification by reverse transcriptases, a problem often encountered in one-step RT-PCR. The buffer ensures specific primer annealing over a wide range of temperatures and Mg²⁺ concentrations; providing robust and highly efficient RT-PCR from any RNA template. The QIAGEN OneStep RT-PCR Kit includes Q-Solution, an innovative additive that modifies the melting behavior of nucleic acids. Q-Solution facilitates reverse transcription and amplification of templates with a high GC content or a high degree of secondary structure (see figure " RT-PCR of GC-rich template"). Using Q-Solution simplifies optimization of RT-PCR for difficult templates. The QIAGEN OneStep RT-PCR Kit includes everything required for faster and easier RT-PCR for even the most sensitive applications (see table).

Reliable one-step RT-PCR results
QIAGEN OneStep RT-PCR Kit contents	Features
HotStarTaq DNA Polymerase	Highly specific products
Sensiscript and Omniscript Reverse Transcriptases	Wide range of RNA amounts (1 pg – 2 µg) High sensitivity
OneStep RT-PCR Buffer	Minimal optimization needed No inhibition of PCR by reverse transcriptases Inhibition of RNases
Q-Solution	Facilitates amplification of GC-rich templates

See figures

Highly specific RT-PCR using low amounts of template.

Efficient detection of viral RNA.

Increased specific primer annealing.

Influence of annealing temperature on specificity.

RT-PCR of GC-rich template.

Procedure

The QIAGEN OneStep RT-PCR Kit allows fast and easy RT-PCR setup. Whatever the application — virus detection, molecular diagnostics research, or gene expression analysis — just mix all components together in one tube and start the thermal-cycler program (see flowchart " OneStep RT-PCR procedure"). The reaction mixture contains all of the reagents required for both reverse transcription and PCR; nothing needs to be added once the reaction has been started (see table).

See figures

OneStep RT-PCR procedure.

Applications

The QIAGEN OneStep RT-PCR Kit is suitable for RT-PCR applications such as:

Virus detection
Single-cell RT-PCR
Gene-expression analysis

Supporting data and figures

OneStep RT-PCR procedure.

The QIAGEN OneStep RT-PCR Kit allows fast and easy RT-PCR setup. Whatever the application — virus detection, molecular diagnostics research, or gene expression analysis— just mix all components together in one tube and start the thermal-cycler program. The reaction mixture contains all of the reagents required for both reverse transcription and PCR; nothing needs to be added once the reaction has been started.

Specifications

Features	Specifications
Applications	Gene-expression analysis, virus detection
Mastermix	No
Enzyme activity	Reverse transcription, 5' -> 3' exonuclease activity
Reaction type	One-step RT-PCR
Real-time or endpoint	Endpoint
Sample/target type	RNA template
Single or multiplex	Single
With/without hotstart	With hotstart

Resources

Download Safety Data Sheets for QIAGEN product components.

Second edition — innovative tools

Addressing critical factors and new solutions

For fast and efficient one-step RT-PCR

Publications

Up-regulation of cyclooxygenase-2 expression is involved in R(+)-methanandamide-induced apoptotic death of human neuroglioma cells.

Hinz B; Ramer R; Eichele K; Weinzierl U; Brune K;

Mol Pharmacol; 2004; 66 (6):1643-51 2004 Sep 10 PMID:15361550

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA.

Kiehart DP; Franke JD; Chee MK; Montague RA; Chen TL; Roote J; Ashburner M;

Genetics; 2004; 168 (3):1337-52 2004 Nov PMID:15579689

Instability of the restriction fragment length polymorphism pattern of open reading frame 5 of porcine reproductive and respiratory syndrome virus during sequential pig-to-pig passages.

Cha SH; Chang CC; Yoon KJ;

J Clin Microbiol; 2004; 42 (10):4462-7 2004 Oct PMID:15472294

Expression of growth differentiation factor 9 messenger RNA in porcine growing and preovulatory ovarian follicles.

Prochazka R; Nemcova L; Nagyova E; Kanka J;

Biol Reprod; 2004; 71 (4):1290-5 2004 Jun 9 PMID:15189836

Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus.

Lee CW; Senne DA; Suarez DL;

J Virol; 2004; 78 (15):8372-81 2004 Aug PMID:15254209

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

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

No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA Polymerase, QIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.

FAQ ID -204

Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).

FAQ ID -961

No. The 5x OneStep RT-PCR Buffer does not contain BSA.

FAQ ID -326

No, the initial activation time of 15 minutes at 95°C is crucial. Enzyme activation will be incomplete when using shorter activation times, resulting in inefficient PCR product amplification.

FAQ ID -565