Journal
PLOS ONE
Volume 9, Issue 3, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0093031
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Funding
- REBIRTH-Cluster of Excellence (Deutsche Forschungsgemeinschaft)
- German Centre for Infection Research (DZIF, TTU Hepatitis)
- Else Kroner-Fresenius-Stiftung (FWRV) [2010_A49]
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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 Delta CT calculation can be extended (e-Delta CT) by replacing the CT of a single RG in Delta CT 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-Delta CT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.
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