title: Variability of the reverse transcription step: practical implications.
abstract: BACKGROUND
  The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability.
  
  
  METHODS
  We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based reverse transcription real-time quantitative PCR (RT-qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same real-time quantitative PCR (qPCR) assay.
  
  
  RESULTS
  RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed.
  
  
  CONCLUSIONS
  Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.
authors.affiliation: Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK; stephen.bustin@anglia.ac.uk.
authors.forename: Stephen
authors.initials: S
authors.lastname: Bustin
authors.affiliation: Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK;
authors.forename: Harvinder S
authors.initials: HS
authors.lastname: Dhillon
authors.affiliation: Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK;
authors.forename: Sara
authors.initials: S
authors.lastname: Kirvell
authors.affiliation: Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK;
authors.forename: Christina
authors.initials: C
authors.lastname: Greenwood
authors.affiliation: Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK;
authors.forename: Michael
authors.initials: M
authors.lastname: Parker
authors.affiliation: Shipley Consulting, LLC, Austin, TX;
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Shipley
authors.affiliation: Institute of Population Health, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.
authors.forename: Tania
authors.initials: T
authors.lastname: Nolan
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012194:RNA-Directed DNA Polymerase