Stephen Bustin1, Harvinder S Dhillon2, Sara Kirvell2, Christina Greenwood2, Michael Parker2, Gregory L Shipley3, Tania Nolan4. 1. Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK; stephen.bustin@anglia.ac.uk. 2. Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, UK; 3. Shipley Consulting, LLC, Austin, TX; 4. Institute of Population Health, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.
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.
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: Philipp Kaiser; Sunil K Joshi; Peggy Kim; Peilin Li; Hongbing Liu; Andrew P Rice; Joseph K Wong; Steven A Yukl Journal: J Virol Methods Date: 2016-12-27 Impact factor: 2.014
Authors: Helmi Pett; Bronner P Gonçalves; Alassane Dicko; Issa Nébié; Alfred B Tiono; Kjerstin Lanke; John Bradley; Ingrid Chen; Halimatou Diawara; Almahamoudou Mahamar; Harouna M Soumare; Sekou F Traore; Ibrahima Baber; Sodiomon B Sirima; Robert Sauerwein; Joelle Brown; Roly Gosling; Ingrid Felger; Chris Drakeley; Teun Bousema Journal: Malar J Date: 2016-11-08 Impact factor: 2.979