## Additional file 2.
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 C_{t}. C) The raw crossing threshold (C_{t}) 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 C_{t }data for all three reference genes (Atp5b, Canx, Rpl13a) for each sample. (E) The geometric mean is transformed into a ΔC_{t }value, thus expressing each sample with respect to the least expressed sample. To
do this, the C_{t }of the least expressed sample is subtracted from the current sample C_{t}, giving a negative value for each sample. The least expressed sample will have a
value of zero. (F) The ΔC_{t }is calculated for the Bdnf data. (G) The ΔΔC_{t }is calculated, by performing the function (C_{t }sample - C_{t }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 -ΔΔC_{t }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.
Format: PPT Size: 131KB Download file This file can be viewed with: Microsoft PowerPoint Viewer Benn |