Variability of the Reverse Transcription Step: Practical Implications

Stephen Bustin2,1, Harvinder Dhillon2, Sara Kirvell2, Christina Greenwood2, Michael Parker2, Gregory Shipley4,1, Tania Nolan1,3
1The Gene Team, United Kingdom; 
2Postgraduate Medical Institute, Anglia Ruskin University, Cambridge, UK; 
3Institute of Population Health, University of Manchester, Manchester UK; 
4Shipley Consulting, LLC, Austin, Texas, USA

The conversion of RNA to cDNA using reverse transcription is a necessary first step for many molecular biology applicatons. While RT efficiency is known to be variable, little attention has been paid to the practical implications of the variability. Several mRNA targets were quantified in RNA samples of varying quantity and quality after RT using commercial reverse transcriptases for cDNA synthesis. An analysis of the relative yield of the markers across the samplesdemonstrated that RT efficiency is enyzme, sample and RNA concentration dependent, resulting in variable correlations between markers in the same sample. This variability translated into relative mRNA expression differences that generally varied between 2- and 3- fold, although higher extremes were observed. These data demonstrate that the inherent variability of the technique is sufficient to call into question the validity ofmany published data sets. Variability can be minimised by selecting the appropriate RT enzyme, concentration of RNA and sufficient characterisation of the individual assay.

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