Comparison of the ( SavrgE )Ct, ( PavrgE )Ctand ΔCt methods to quantify gene expression regulation. cDNAs were prepared from RNA extracted from fibroblastic cells induced or not by TGF-β treatment, as described in the Materials and methods. Expression as determined from the mRNA levels of the plasminogen activator inhibitor 1 (PAI-1), fibronectin (FN) and connective tissue growth factor (CTGF) genes were normalized to those of the ribosomal L27 protein, used as an invariant internal reference. Normalized gene expression was calculated using the (SavrgE)Ct, (PavrgE)Ctor the ΔCt methods, as indicated, using either the complete set of 10 replicate assays (top histograms), or using just three measurements (first three assays of the series, bottom histograms). Error bars represent standard deviations on the normalized ratio. A t-test was performed on the normalized gene expression to check whether the expression were statistically different between the induced and the non-induced state (* = p < 0.05, ** = p < 0.001, # = p > 0.1).