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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Reverse transcription (RT) is essential for converting RNA to cDNA in research and diagnostics.
  • The variability in RT efficiency has significant, often overlooked, practical implications.

Purpose of the Study:

  • To investigate the reproducibility of the RT step using commercial reverse transcriptases.
  • To assess the impact of RNA quality and concentration on RT variability.
  • To evaluate the correlation between mRNA targets amplified from the same sample.

Main Methods:

  • Utilized commercial reverse transcriptases and RNA samples of varying quality and concentration.
  • Quantified multiple mRNA targets using singleplex SYBR Green I or dualplex probe-based RT-qPCR.
  • Calculated correlations between quantification cycles (Cqs) of mRNA targets within the same qPCR assay.

Main Results:

  • RT efficiency is dependent on enzyme, sample, RNA concentration, and assay type.
  • This variability leads to inconsistent correlations between mRNA targets from the same sample.
  • Observed relative mRNA expression level variations typically ranging from 2- to 3-fold, with higher variations also noted.

Conclusions:

  • The inherent variability in the RT step challenges the validity of many published cDNA quantification studies.
  • Minimizing variability requires careful selection of reverse transcriptase and high RNA concentrations.
  • Characterizing assay variability through multiple RT replicates is recommended for robust results.