Pfaffl analysis. Example of Pfaffl calculation on serum miRNA RT-qPCR data performed in Microsoft Excel 2010. In this experiment, serum abundance of miR-206 was compared between C57 (wild type control) and mdx (dystrophic) mice aged 16 weeks (n = 4). (a) PCR efficiencies determined experimentally for each assay. If PCR efficiency is unknown it can be approximated as 2 (i.e. 100%). (b) Cq data from RT-qPCR run. (c) Pfaffl transformation converts Cq values into amounts in arbitrary units [equation in E4, =POWER($B$2,-B4)]. (d) Gene-of-interest (miR-206) abundance is normalized by dividing the Pfaffl transformed quantity by that of the reference control (cel-miR-39) (equation in H4, =F4/E4). (e) The mean normalized quantity of the control is calculated [equation in H15, =AVERAGE(H4-H7)]. (f) Dividing by the mean of the control group returns the control to a value of one so that fold changes between experimental groups can be visualized more easily (equation in J4, =H4/$H$15). (g) The mean and standard error of the mean (SEM) are calculated for the two experimental groups. (h) A t-test performed in Microsoft Excel indicates a highly statistically significant difference between the groups [equation in D15, =TTEST(J4-J7,J8-J-11,2,2)]. (i) Graph of serum miRNA abundance data. Values are mean +/- SEM, ***P < 0.001.