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.