AAT Bioquest

How do I optimize my reverse transcription process?

Posted March 1, 2023


There are five main factors you need to consider in order to optimize your reverse transcription process and obtain high yields of low copy messages.  

  1. Choose the appropriate RT Primer - There are three RT primers you can choose from – oligo (dT) primers, random primers and gene specific primers. Choose Oligo (dT) primers for obtaining full length copies of mRNA. Random primers are a better option for transcribing 5’ ends of long genes. However, the cDNAs may not be full length copies of the entire genome. Choose gene specific primers if you are performing a one-step RT-PCR. The RT primer is also your reverse primer for the PCR step. 
  1. Remove gDNA - False positives in the final PCR are often caused by genomic contamination in the RNA. Removing gDNA can minimize this error. gDNA can be removed via DNase treatments during an RNA prep. 
  1. Check RNA Integrity - Overall RNA quality and bath-to-batch consistency in RNA quality are critical for reliable results. Run an agarose gel to check the quality of the rRNA bands. Undamaged eukaryotic RNA should show 28s and 18s rRNA.
  1. Remove Secondary Structure in RNA - Secondary structure in RNA can cause the RT enzyme to stop or fall off the template when it approaches loop structures in the RNA. This can be a problem if you are transcribing full length RNA. A 5-minute denaturation at 65°C can help relax the RNA and ensures that it will be completely single-stranded before the reaction. Another option for removing RNA secondary structure is to use an RT enzyme that allows synthesis to occur at a temperature higher than standard RT. 
  1. Decide between One-Step RT-PCR and Two-Step RT-PCR - The choice between one-step RT-PCR and two-step RT-PCR comes down to personal preference. Understanding the advantages of each can help you determine which to choose for your assay. Removal of a sample transfer step in one-step RT-PCR eliminates a potential source for contamination. Moreover, one-step RT-PCR has higher sensitivity because the primer is gene-specific. Two-step RT-PCR allows you to convert a large batch of RNA to cDNA, which can then be aliquoted into many reactions. It also allows for a larger choice in primers. Another advantage of two-step RT-PCR is that you can use the same RT batch for analyzing multiple genes, thus eliminating potential variation when comparing different genes from the same source. 
Additional resources

Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors

Reverse Transcription PCR (RT-PCR)

Helixyte™ Green *20X Aqueous PCR Solution*