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QIAGEN OneStep Ahead RT-PCR Kit

For faster one-step RT-PCR with high sensitivity, specificity and fidelity
  • Unique two-phase hot-start procedure for room-temperature setup
  • Dedicated enzyme mix for increased specificity, sensitivity and fidelity
  • Faster than ever 1-hour cycling protocol
  • Visual pipetting control, resulting in fewer pipetting errors
  • Duplex capability, enabling inclusion of internal control or reference gene
The QIAGEN One-Step Ahead RT-PCR Kit contains a blend of Sensiscript and Omniscript Reverse Transcriptases, a well-balanced combination of Taq DNA Polymerases and a proofreading enzyme. Heat-mediated activation of these enzymes enables reaction setup at room temperature. The easy one-tube setup and optimized components enable higher sensitivity and successful results. Additional tracing dyes allow the loading process to be monitored, minimizing pipetting errors.
Cat No./ID: 220211
QIAGEN OneStep Ahead RT-PCR Kit (50)
S Fr.248.00
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6 vials for 50 reactions: 1 x 500 µl OneStep Ahead RT-PCR Master Mix, 1 x 50 µl OneStep Ahead RT Mix, 1 x 200 µl Template Tracer, 1 x 50 µl Master Mix Tracer, 1 x 1.9 ml water, 1 x 400 µl Q-Solution
Cat No./ID: 220213
QIAGEN OneStep Ahead RT-PCR Kit (200)
S Fr.844.00
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8 vials for 200 reactions: 2 x 1 ml OneStep Ahead RT-PCR Master Mix, 1 x 200 µl OneStep Ahead RT Mix, 1 x 200 µl Template Tracer, 1 x 50 µl Master Mix Tracer, 2 x 1.9 ml water, 1 x 2 ml Q-Solution
Cat No./ID: 220216
QIAGEN OneStep Ahead RT-PCR Kit (2000)
S Fr.7,427.00
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75 vials for 2000 x 25 µl reactions: 20 x 1 ml OneStep Ahead RT-PCR Master Mix, 10 x 200 µl OneStep Ahead RT Mix, 10 x 200 µl Template Tracer, 10 x 50 µl Master Mix Tracer, 20 x 1.9 ml water, 5 x 2 ml Q-Solution
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Superior stability after reaction setup at room temperature
HeLa total RNA (10 and 1 pg) was used as a template for amplification of ACTB in triplicates, according to suppliers’ instructions. Reactions were either set up on ice or left at room temperature for the times indicated before analysis on a 2% agarose gel. Distinct, gene-specific bands are observed with the QIAGEN OneStep Ahead RT-PCR Kit even after a 2 h incubation at room temperature before cycling (blue arrow), whereas reactions performed with kits from competitors deteriorate as time progresses. Gene-specific bands appear significantly weaker, if present at all, while primer-dimers (red arrows) become more prominent.
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Amplification of GC-rich amplicons
HeLa total RNA (100 ng) was used as template for amplification of TNFRI (581 bp) with a GC ratio of 67.1%. Reactions were performed in triplicate, according to the suppliers’ instructions. Green arrows indicate specific product. Q-Solution was used for the QIAGEN reactions.
2
Amplification of long amplicons
HeLa total RNA (100 and 10 ng) was used as a template for amplification of RANBP2 (3 kbp).
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NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
Potassium ions (K+) bind to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as ammonium ion and as ammonia under thermal‐cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing 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.
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Optional pipetting control
The kit comes with optional pipetting controls. The Master Mix tracer is an inert orange dye that can be added to the Master Mix. The Template Tracer is an inert blue dye that can be added to the template. When adding the template to the Master Mix, the color turns green, providing a visual indication of correct pipetting. In addition, both dyes allow tracking during gel electrophoresis. The dyes run at about 50 bp (orange) and 4000 bp (blue) on a 1% agarose gel.
5
Superior sensitivity
Indicated amounts of HeLa total RNA (in pg) were used as template for amplification of GAPDH (831 bp) and ACTB (295 bp) in duplicate, according to the suppliers’ instructions. Green arrows indicate specific product, red arrows indicate primer‐dimers. Analysis was performed using the QIAxcel.
6
Superior specificity
Indicated amounts of HeLa total RNA (in ng) were used as template for amplification of EIF2B4 (107 bp) in duplicate, according to the suppliers’ instructions. Green arrows indicate specific product, red arrows indicate primer‐dimers, purple arrows indicate nonspecific amplification products. Analysis was performed using the QIAxcel.
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OneStep Ahead RT-PCR procedure
The QIAGEN OneStep Ahead RT-PCR Kit allows fast and easy RT-PCR setup whatever your application - virus detection, molecular diagnostics research or gene expression - just mix all components together in one tube and start your 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. As a visual pipetting control, you can add the optional orange Master Mix Tracer and blue Template Tracer.
Performance
The QIAGEN OneStep Ahead 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 “Superior sensitivity” and “Superior specificity”).

For the reverse-transcription step, both Omniscript and Sensiscript are functionalized with an RT-blocker, which keeps them in an inactive state and prevents nonspecific transcription at ambient temperature. When the reaction is heated to 50°C, the RT-blocker dissociates from the RT enzymes, rendering them fully active.

The PCR amplification step is catalyzed by a well-balanced blend of three different DNA polymerases. QuantiNova DNA Polymerase is provided in an antibody-mediated, inactive state and has no enzymatic activity at ambient temperature. Activation of this enzyme occurs after a 5-minute incubation step at 95ºC, allowing highly specific amplification from the first cycle. HotStarTaq DNA Polymerase activation occurs gradually and ensures high yields of the PCR product. The high-fidelity proofreading DNA Polymerase with 3'→5' exonuclease activity is also heat-activated by the initial 5-minute incubation step at 95°C, ensuring superior amplification accuracy and processivity. The innovative, dual-cation PCR buffer provided in the master mix ensures high yields of specific PCR products over a wide range of annealing temperatures. 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 “Amplification of GC‐rich amplicons”).

Reaction setup at room temperature adds convenience and facilitates use of the kit in automated workflows (see figure “Superior stability after reaction setup at room temperature”). The master mix includes buffer, dNTPs and DNA polymerases. The RT-Mix, which includes the reverse transcription enzymes as well as the RNase-inhibitor, is provided separately to allow for minus RT reactions. To set up the reaction, simply add the primers, target RNA and RT-Mix to the master mix.

Integrated control features prevent artifacts and ensure reliable results. The optional tracking system, comprising a Master Mix Tracer and Template Tracer for visual identification of correct pipetting, minimizes errors during setup (see figure “Optional pipetting control”). The chemistry is optimized for duplex PCR to enable co-amplification of an internal positive control with every reaction. The included RNase inhibitor prevents RNA decay caused by accidental RNase contamination.
Principle
The QIAGEN OneStep Ahead RT-PCR Kit is designed for easy and sensitive one-step RT-PCR using any RNA template. The enzyme mix is specially formulated 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. After reverse transcription, reactions are heated to 95°C for 5 minutes to activate the DNA polymerases and to 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.

The optimal primer annealing temperature is dependent on the base composition, primer concentration and ionic reaction environment. QIAGEN PCR Buffers contain both K+ and NH4+ to ensure high yields of specific PCR products over a wide range of annealing temperatures (see figure “NH4+ and K+ cations in QIAGEN PCR buffers increase 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+. The QIAGEN OneStep Ahead 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 Ahead RT-PCR Kit includes Q-Solution, an additive that modifies the melting behavior of nucleic acids and facilitates reverse transcription and amplification of difficult templates, including those with a high GC content or a high degree of secondary structure. The QIAGEN OneStep Ahead RT-PCR Kit includes everything you need for faster and easier RT-PCR for even the most sensitive applications (see table).
Reliable one-step RT-PCR results
QIAGEN OneStep Ahead RT-PCR  Features  Benefits
Sensiscript and Omniscript reverse transcriptases Wide range of RNA amounts (0.1 pg – 2 µg)
High sensitivity
Heat-mediated activation
Convenient room-temperature setup
Applicable to automated workflows
QuantiNova HotStarTaq, hot-start high-fidelity Polymerase Highly specific products
Heat-mediated activation
Increased fidelity
Short cycling time
Convenient room-temperature setup
High sequence accuracy
Amplification of long targets (<4 kb)
Fast cycling time ~1 h
Master mix with DNA polymerases, Buffers, dNTPs Proprietary dual-cation buffer system
Optimized for duplex PCR
Minimal optimization needed
Convenient reaction setup
No inhibition of PCR by reverse transcriptases
Increased confidence due to optional inclusion of positive control with every reaction
RNase inhibitor Inhibition of RNases RNA decay protection
Q-Solution Facilitates amplification of GC-rich templates Better and more reliable performance
Optional pipetting control Inert orange and blue dyes
Run-on gel at 50 bp (orange) and 4000 bp (blue)
Highest confidence by minimizing pipetting errors
Also serves as a gel tracking dye

Procedure
The QIAGEN OneStep Ahead RT-PCR Kit allows fast and easy RT-PCR setup whatever the application – virus detection, molecular diagnostics research or gene expression analysis – just add the RT-enzyme mix, primers and template to the Master Mix in one tube and start the thermal-cycler program (see figure “OneStep Ahead RT-PCR procedure”).. Reaction setup can conveniently be done at room temperature and the optional master mix and template tracers minimize pipetting errors. The reaction mixture contains all of the reagents required for both reverse transcription and PCR (see table).
Applications
The QIAGEN OneStep Ahead RT-PCR Kit is suitable for RT-PCR applications such as:
  • Virus detection
  • Gene expression analysis
Features
Specifications
Applications Gene expression analysis, virus detection
Applications Gene expression analysis, virus detection
Enzyme activity Reverse transcription, 5' -> 3' exonuclease activity
Enzyme activity Reverse transcription, 5' -> 3' exonuclease activity
Mastermix Yes
Mastermix Yes
Reaction type One-step RT-PCR
Reaction type One-step RT-PCR
Real-time or endpoint End-point
Real-time or endpoint End-point
Sample/target type RNA template
Sample/target type RNA template
Single or multiplex Single/duplex
Single or multiplex Single/duplex
With/without hotstart With hot-start
With/without hotstart With hot-start
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