Additional file 3.
GeNorm analyses to identify optimal reference genes in the cerebellum. (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. Reference genes tested were 18S (18S ribosomal RNA subunit), Actb (beta-actin), Atp5b (ATP synthase subunit 5b), B2m (beta-2 microglobulin), Canx (calnexin), Cyc1 (cyclin D1), Eif4a2 (eukaryotic initiation factor 4a2), Gapdh (glyceraldehyde-3-phosphate dehydrogenase), Rpl13a (ribosomal protein L13a), Sdha (succinate dehydrogenase complex, subunit A), Ubc (ubiquitin C) and Yhwaz (phospholipaase A2). (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 (ie 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 most stably expressed genes on the left with the inclusion of a 4th gene etc, moving to the right. This measure is known as pairwise variation (V), the values of which are indicated above each bar. A V score of below 0.15 is the target.
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Benn et al. Molecular Neurodegeneration 2008 3:17 doi:10.1186/1750-1326-3-17