Chunk #76 — ONLINE METHODS — Normalization of Gene Expression and Adjustment for Covariates — Technical validation of normalized gene expression levels using qPCR
Some of these genes showed increased expression and others showed decreased expression between cases and controls in the Pitt cohort, and many have been reported to be similarly altered in other cohorts of SCZ subjects. After selection of uniquely-mapping primers (approximately 20 bp for each of forward and reverse strand), qPCR was performed for each of these 13 genes and mRNA levels were normalized to the expression of ACTB, PPIA, and GAPDH, yielding “expression ratios” calculated using CTs (i.e., the PCR cycle threshold). The Pearson correlation between these expression ratios and the voom-normalized log(CPM) levels for the same subjects was greater than 0.5 for 9 of the 13 genes (Supplementary Fig. 3A); for an additional 3 genes, it was between 0.1 and 0.3, and only for one gene (HIVEP2) was the correlation negative. The correspondence between estimates is notable because of the different measurement methodologies and because, while the samples came from the same subject and brain region, they were drawn independently for the qPCR and RNA-seq experiments. We thus conclude that the genome-wide RNA-seq-based quantification provides good estimates of
