Variability of the Reverse Transcription Step: Practical Implications
| Autor: | Gregory L. Shipley, Stephen A. Bustin, Sara Kirvell, Christina Greenwood, Tania Nolan, Harvinder S. Dhillon, Mike Parker |
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| Rok vydání: | 2015 |
| Předmět: |
Messenger RNA
DNA Complementary Reverse Transcriptase Polymerase Chain Reaction Biochemistry (medical) Clinical Biochemistry Glyceraldehyde-3-Phosphate Dehydrogenases Reproducibility of Results RNA RNA-Directed DNA Polymerase Biology Sensitivity and Specificity Molecular biology Reverse transcriptase chemistry.chemical_compound Real-time polymerase chain reaction Molecular Diagnostic Techniques chemistry Complementary DNA SYBR Green I RNA Messenger Practical implications DNA |
| Zdroj: | Clinical Chemistry. 61:202-212 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1373/clinchem.2014.230615 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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