QIAGEN OneStep Ahead RT-PCR Kit
For faster one-step RT-PCR with high sensitivity, specificity and fidelity
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.
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.
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).
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).
The QIAGEN OneStep Ahead RT-PCR Kit is suitable for RT-PCR applications such as:
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