QIAGEN OneStep RT-PCR Kit

For highly sensitive and specific one-step RT-PCR
  • 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
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.
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QIAGEN OneStep RT-PCR Kit (25)
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)
 210210
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QIAGEN OneStep RT-PCR Kit (100)
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)
 210212
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QIAGEN OneStep RT-PCR Kit (1000)
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)
 210215
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The QIAGEN OneStep RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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OneStep RT-PCR procedure.|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.|Increased specific primer annealing.|
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.|One-step RT-PCR was carried out using the indicated amounts of total RNA from HeLa cells and primers specific for α-catenin, amplifying a 690 bp product. All reactions were carried out following suppliers' instructions. M: 100 bp ladder.|A 336 bp fragment of F-gene mRNA was reverse-transcribed and amplified from Sendai virus RNA isolated from persistently infected Vero cells. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit and the indicated number of viral genome copies. M: markers. (Data kindly provided by H. Rausch, Max Planck Institute for Biochemistry, Martinsried, Germany as part of the project "Experimental control of virological work at safety levels 2 and 3 in Bavaria," supported by the Bavarian Ministry of the Environment.)|One-step RT-PCR was performed using kits from the indicated suppliers over a range of annealing temperatures. A 1289 bp fragment from the human RCC1 gene was reverse transcribed and amplified from Hela RNA (arrow). M: markers. High levels of specific amplification without optimization were observed only with the QIAGEN OneStep RT-PCR Kit.|A 439 bp fragment of transcription factor TAFII100 mRNA (GC content: 75%) was reverse-transcribed and amplified from HeLa-cell total RNA. Reactions were prepared using the QIAGEN OneStep RT-PCR Kit with (+) or without (–) Q-Solution. Q-Solution ensures specific amplification of GC-rich templates. M: markers.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specific primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific-to- nonspecific primer-template binding over a wide temperature range.|
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 "Highly specific RT-PCR using low amounts of template" and "Efficient detection of viral RNA"). 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 "Influence of annealing temperature on specificity"). 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 "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 contentsFeatures
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
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).

Applications

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

  • Virus detection
  • Single-cell RT-PCR
  • Gene-expression analysis
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Feature
Specifications
Applications Gene-expression analysis, virus detection
Enzyme activity Reverse transcription, 5' -> 3' exonuclease activity
Mastermix No
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

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Kit Handbooks
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(EN) - QIAGEN OneStep RT-PCR Kit Handbook
For fast and efficient one-step RT-PCR
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