title: An extended ΔCT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells.
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.
authors.affiliation: REBIRTH Research Group "Hepatic Cell Therapy", Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Gesa
authors.initials: G
authors.lastname: Riedel
authors.affiliation: REBIRTH Research Group "Hepatic Cell Therapy", Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Urda
authors.initials: U
authors.lastname: Rüdrich
authors.affiliation: REBIRTH Research Group "Hepatic Cell Therapy", Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Nora
authors.initials: N
authors.lastname: Fekete-Drimusz
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Manns
authors.affiliation: Regenerative Medicine & Experimental Surgery (ReMediES), Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors.forename: Florian W R
authors.initials: FW
authors.lastname: Vondran
authors.affiliation: REBIRTH Research Group "Hepatic Cell Therapy", Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Bock
mesh_terms: D002460:Cell Line; D015870:Gene Expression; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction