Shedding light: The importance of reverse transcription efficiency standards in data interpretation

Publication date: March 2019Source: Biomolecular Detection and Quantification, Volume 17Author(s): Jessica Schwaber, Stacey Andersen, Lars NielsenAbstractThe RNA-to-cDNA conversion step in transcriptomics experiments is widely recognised as inefficient and variable, casting doubt on the ability to do quantitative transcriptomics analyses. Multiple studies have focused on ways to optimise this process, resulting in contradictory recommendations. Here we explore the problem of reverse transcription efficiency using digital PCR and the RT method’s impact on subsequent data analysis. Using synthetic RNA standards, an example experiment is presented, outlining a method to (1) determine relevant efficiency and variability values and then to (2) incorporate this information into downstream analyses as a way to improve the accuracy of quantitative transcriptomics experiments.
Source: Biomolecular Detection and Quantification - Category: Molecular Biology Source Type: research