Additional file 2.
Example worksheet illustrating 2-ΔΔCt analysis process. Shown is an Excel worksheet illustrating the process of relative expression analysis. In the worksheet are numbers in blue which illustrate each step. (A) Crossing threshold data can be imported into Excel from real-time PCR platforms (shown are the Bdnf data only for simplicity). (B) Each sample has been run in triplicate and the means of each sample is calculated. Standard deviations should be checked at this point and should be within 1 Ct. C) The raw crossing threshold (Ct) data can be used to plot a graph of the means for each genotype. Shown are means (Avg), standard deviations (SD) and standard error of the mean (SEM) for WT (wild-type) and TG (R6/2) mice (n = 7/genotype). The means are used to generate the graphs, and error bars are SEM. (D) The geometric mean (Geo, or GEOMEAN) is calculated using the raw Ct data for all three reference genes (Atp5b, Canx, Rpl13a) for each sample. (E) The geometric mean is transformed into a ΔCt value, thus expressing each sample with respect to the least expressed sample. To do this, the Ct of the least expressed sample is subtracted from the current sample Ct, giving a negative value for each sample. The least expressed sample will have a value of zero. (F) The ΔCt is calculated for the Bdnf data. (G) The ΔΔCt is calculated, by performing the function (Ct sample - Ct reference), which will give both positive and negative values. (H) To transform the ΔΔCt values into positive integers that represent the expression levels, use the Excel POWER function, entering 2 as the number and -ΔΔCt as the power. The negative sign is necessary in this context. (J) The relative expression levels for each sample can then be used to calculate the means (Avg), standard deviation (SD) and standard error of the mean (SEM) for each genotype. These data are used to generate graphs showing expression ratios for target genes. (K) In addition, the expression ratios can be used as a substrate for statistical analyses such as a Student's t-test.
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Benn et al. Molecular Neurodegeneration 2008 3:17 doi:10.1186/1750-1326-3-17