GeNorm analyses to identify optimal reference genes in the striatum. (A) Raw crossing threshold (Ct) data for a panel of 12 potential references from the geNorm kit in wild-type (open bars) and R6/2 (filled bars) mice. (B) Raw Ct data was subjected to analysis with the geNorm applet which automatically calculates the gene-stability measure M, which is an average pairwise variation of a particular gene with all other control genes. Therefore, genes with the lowest M value have the most stable expression, in this case across genotypes (i.e. comparing wild-type and R6/2 mice). Expression stability is plotted for each of the potential reference genes, progressing from the least stable genes with a higher M value to the most stable genes with a lower M value. (C) In order to measure expression levels accurately, normalization by multiple housekeeping genes is optimal. The graph illustrates the levels of variation in average reference gene stability with the sequential addition of each reference gene to the equation, starting with the three most stably expressed genes on the left with the inclusion of a 4th gene and so on, moving to the right. This measure is known as pairwise variation (V), the values of which are indicated above each bar, with a score of <0.15 as a target.
Benn et al. Molecular Neurodegeneration 2008 3:17 doi:10.1186/1750-1326-3-17