Table 5.

Reference target stability values (M and CV)

PFC
Reference Target	M	CV
CYC1	0.4716	0.1812
GAPDH	0.6866	0.2651
PPIA	0.4709	0.2495
Average	0.5430	0.2319
Hippocampus
Reference Target	M	CV
CYC1	0.5128	0.2437
GAPDH	0.5878	0.2331
PPIA	0.4601	0.1525
Average	0.5202	0.2098

Reference target stability values (M and CV) determined for the combination of the reference targets GAPDH, CYC1, and PPIA in human brain samples from the PFC and hippocampus. Expression stability of reference genes in human brain samples was calculated by geNorm algorithm using qBasePLUS software (Biogazelle) as described in our previous publication (Fuchsova et al., 2015). GeNorm analysis was performed on the gene expression data from PFC and hippocampal postmortem samples obtained from all depressed subjects and matched nonpsychiatric controls. The gene stability measure M is defined as the average pairwise variation of a particular gene with all other control genes (Vandesompele et al., 2002). Genes with lowest values have the most stable expression (optimal geNorm M≤0.5). To assess that the genes with the lowest M values have indeed the most stable expression, the gene-specific variation of each control gene is determined as the variation coefficient (CV) of the expression levels after normalization. Mean CV values equal or lower than 0.2 are typically observed for stably expressed reference genes in relatively homogenous sample panels. Normalization to a single control gene can lead to erroneous normalization. For this reason, in our previous publication the optimum number of three reference genes required for adequate data normalization in this experimental situation was calculated using geNorm (Fuchsova et al., 2015). Thus, in our experiments in human postmortem PFC and hippocampal samples, the optimal normalization factor was calculated as the geometric mean of reference targets PPIA, CYC1, and GAPDH.