An Extended ΔCT-Method Facilitating Normalisation with Multiple Reference Genes Suited for Quantitative RT-PCR Analyses of Human Hepatocyte-Like Cells

<div><p>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 <i>in vitro</i> 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 <i>PSMB6, MDH1</i> and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells <i>in vitro</i>. 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.</p></div

e-ΔCT-method and representative calculations of changes of target gene expression levels (expressed as fold change).

<p>(A) Calculation of e-ΔCT, an extension of the ΔCT-method by first calculating the mean CT-value of multiple RG included for each sample. (B) Depiction of data processing after geNorm-/e-ΔCT-calculations of sample-specific G-values to calculate fold changes and their significance (p-values) between two experimental settings, each consisting of three fully independent samples. (C) Example (Huh-7, without vs. with ActD) comparing fold changes in gene expression levels calculated by geNorm (grey) and e-ΔCT (black) using: GAPDH, the most common RG, the AS best ranking PSMB6, MDH1 and ACTB, and inclusion of two (PSMB6 and MDH1; P_M) or three (PSMB6, MDH1 and ACTB; P_M_A) RG. (D) Examples (primary hepatocytes, 0 vs. 24 hrs) comparing use of most stable RG of the subgroup (RG group, i.e. PH) vs. overall RG (RG all, i.e. AS) via e-ΔCT. *: p-values <0.05.</p

Evaluation and ranking of reference genes.

<p>(A) Overall ranking of 22 RG using geNorm, Bestkeeper and Normfinder algorithms and calculation of the cumulative ranking (column 4). (B) geNorm derived M-values as measures for the average pairwise expression stabilities within AS (column 1), and in the subgroups DD (2), CC (3) and PH (4). (C) Box-whisker plots of all CT-values of reference genes and target genes examined. Median (central horizontal line), the 25th and 75th quartile (boxes) and whiskers for the total CT-range are shown.</p