High-Precision PCR


1Two-Step RT-PCR was done using an HCV Armored RNA target in a background of human leukocyte total RNA.  The HCV sequence was from the 5'UTR of the virus and   has moderate secondary structure.  Each reverse transcription sample contained about 1,000 targets based on the manufacturer's estimates and the buffer recommended by the reverse transcriptase supplier was used.  In some cases, reverse transcription was also tested using Invitrogen PCR buffer with 3 mM magnesium.   Reverse transcription was done at 55oC for 30 minutes.  Each RT sample was used to prepare 4 PCR samples.



2The result "±" under non-specific product indicates the presence of a band the size of primer-dimer, but no other non-specific products.


3WarmStart RTx from New England Biolabs is an MMLV-derived transcriptase with RNase H activity that is optimized for LAMP and includes an aptamer for hot start.



4TGIRT is a Group II reverse transcriptase and the manufacturer's protocol is optimized for RNA Seq.  The buffer has extremely high salt concentrations and no specific amplification was observed after RT in that buffer, although adjustments were made for the PCR step.  Low level probe signal was observed after RT in PCR buffer samples containing 1 or 2 units of ThermaStop-RT.

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(TS and TS RT Test Data RNA Transcriptase)