An extended ΔCT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells

PLoS One. 2014 Mar 21;9(3):e93031. doi: 10.1371/journal.pone.0093031. eCollection 2014.

Abstract

Reference genes (RG) as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR) must be stably expressed within the experimental range. A variety of in vitro cell culture settings with primary human hepatocytes, and Huh-7 and HepG2 cell lines, were used to determine candidate RG expression stability in RT-qPCR analyses. Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies PSMB6, MDH1 and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells in vitro. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT) by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG--here identified suited for human hepatocyte-like cells--for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Gene Expression Profiling
  • Gene Expression*
  • Hepatocytes / metabolism*
  • Humans
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards*
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction

Grants and funding

This work was funded by the REBIRTH-Cluster of Excellence (Deutsche Forschungsgemeinschaft), the German Centre for Infection Research (DZIF, TTU Hepatitis) and the Else Kröner-Fresenius-Stiftung (FWRV; 2010_A49). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.