Description
The StableScript™ One-Step RT-qPCR Mix is a highly efficient and sensitive RT-qPCR formulation for DNA and RNA detection using hydrolysis probes. The 10X enzyme mix contains a blend of a new RNAse H minus reverse transcriptase with higher thermostability and inhibitor resistance (StableScript™), a fast-activating hot start DNA polymerase (Phoenix™), and specific enhancers to increase sensitivity and inhibitor resistance. StableScript™ One-Step RT-qPCR Mix is supplied with 4X StableScript™ Reaction Buffer B7720L, containing dNTPs and Magnesium.
StableScript™ One-Step RT-qPCR Mix is a blend of StableScript™ Reverse Transcriptase and Phoenix™ Hot Start Taq DNA Polymerase enzymes. StableScript™ Reverse Transcriptase is engineered for increased thermostability. Increasing the temperature for cDNA synthesis can aid in overcoming challenging RNA targets because higher temperatures help unravel RNA structures, reducing the likelihood of hindering reverse transcription. The Phoenix™ Polymerase is a recombinant, thermostable Taq DNA polymerase complexed with a thermolabile, neutralizing antibody. This fast-activating antibody-mediated hot-start capability enhances the overall specificity, sensitivity, and yield of the PCR reaction by reducing nonspecific amplification and primer-dimer formation prior to cycling. Together with StableScript™, Phoenix™ creates StableScript™ One-Step RT-qPCR Mix, an extremely sensitive RT-qPCR mix: qPCR formulation for detection using hydrolysis probes Robust inhibitor resistance Able to multiplex while maintaining sensitivity and specificity Detects as little as 0.1pg of RNA in 4 plex assay Increased thermostability with optimal cDNA synthesis from 50ºC to 65ºC Stable on benchtop overnight Excellent dynamic range on a variety of assay systems Exceptional lot-to-lot consistency Mix properties Storage temperature: –25°C to –15°C
StableScript™ One Step RT-qPCR reaction setup General precautions against degradation of RNA template should be taken when setting up a reaction, including setting up the reaction with nuclease free water, RNase inhibitor, nuclease free PCR tubes and sterile pipette tips with filter, adding reverse transcriptase last, gentle pipetting, thorough mixing and a quick centrifugation. The following procedure can be used as a guideline. Reactions may need to be optimized individually. The 4X StableScript™ Reaction Buffer should be thaw completely and vortex for 3-5 seconds to mix thoroughly. Quickly spin to collect contents. Prepare the primer/probe mix. A final concentration of 0.4-0.9μM for each primer and 0.1-0.5μM for the probe is recommended. However, the optimal concentration for primers/probes needs to be empirically determined for each assay. Determine the number of reactions to prepare, including No Template Controls (NTCs). Add 10% extra volume to compensate for the pipetting loss. Follow the table below to set up the reaction mix. It is recommended to make a master mix to minimize variations and potential errors. Components Volume/Rxn Final Concentration 4X StableScript™ Reaction Buffer 5μL 1X Primer/Probe Mix XμL Variable 10X StableScript™ Enzyme Mix 2μL 1X RNA Template Up to 2μL 1pg to 1µg total RNA Nuclease-Free Water To a final 20μL reaction volume - Seal the PCR plate and spin briefly to collect contents at the bottom. Thermal Cycling Conditions: Program the cycling conditions are recommended as below. Standard Cycling Program* Steps Temperature Time Cycles Reverse Transcription 55°C 10 min 1 Taq Activation/Initial Denaturation 95°C 3 min 1 Denaturation 95°C 5-10 sec 40 Annealing/Extension* 60°C 30-60 sec *Cycling parameters can be modified (especially the annealing/extension condition) to fit specific assays. Quality control analysis The functionality of the RT-PCR Assay is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The amplification threshold (Ct) of the test lot is compared to a reference lot. Notes: Enzyme components were tested prior to formulation of the master mix and found free of contaminating endonucleases and exonucleases. Enzyme purity was >99% as determined by SDS-PAGE, and negligible E. coli genomic DNA contamination was confirmed by qPCR. Specific activity was verified for each enzyme pre-formulation.
