Figure 2

Coefficients of variation for normalized quantity of IL-1β in serial dilutions of the RT-PCR template. Quantities of IL-1β, HPRT, SDHA and ACTB mRNAs (the R₀ values) were determined in each dilution of the RT-PCR template (six dilutions of total RNA) using all the tested methods of real-time PCR data analysis. The amount of IL-1β mRNA in each dilution was then divided by the relative amount of HPRT, ACTB, or by the normalization factor (NF, geometric mean of HPRT, SDHA and ACTB amounts) of the dilution. Arithmetical mean, standard deviation and coefficient of variation of the normalized IL-1β quantity for each type of normalization (HPRT, ACTB, NF) and each method of real-time PCR data analysis was then calculated. Coefficients of variation (CV) are shown: the first columns are CV values after normalization with the normalization factor, the second columns are CV values after normalization with ACTB, and the third columns are CV values after normalization with HPRT. Methods for real-time PCR data analysis: St. C., relative standard curve; COM, comparative Ct; SCF, sigmoid curve-fitting; DAR iE, DART-PCR with individual E values; DAR aE, DART-PCR with average E values; L&S iE, Liu & Saint-exp with individual E values, L&S aE, Liu & Saint-exp with average E values; LR iE, LinRegPCR (using individual E values); LR-Ct, LinRegPCR combined with Ct (using average E values)