Experimental design and comparison of miRNA expression between samples with and without pre-amplification. A, The same RNA (150 ng) was used in the reverse transcription (RT) and pre-amplification reactions. B and C, correlation plot between raw Ct values for each replicate with (Amp) or without (RT) pre-amplification. D, correlation plot of ΔCt obtained with and without pre-amplification. E, difference in miRNA expression (ΔΔCt) between amplified and non-amplified samples. ΔΔCt values were plotted as the average Ct of non-amplified samples. F, correlation plot between raw Ct values obtained with and without pre-amplification. Data points represented by white dots indicate miRNAs that were only detectable in pre-amplified samples. G and H, correlation plot of Ct values of miRNAs detected only in pre-amplified samples between replicates with and without pre-amplification. Corresponding r and p value determined by linear regression analysis and sample number presented in each plot.