Chunk #75 — ONLINE METHODS — Normalization of Gene Expression and Adjustment for Covariates — Technical validation of normalized gene expression levels using qPCR
The voom-normalized log(CPM) levels provide estimates of true gene expression. To determine if these estimates were precise, we compared their values to independent estimates of gene expression. Studies reporting validation of their RNA quantification typically report “technical validation;” i.e., after extraction from a common source, an RNA pool is measured by the primary quantification tool and the same pool is assessed by a secondary quantification tool, such as qPCR. Technical validation often results in excellent fit between the two methods; yet it avoids other sources of experimental variation involved in extracting RNA from tissue. We take a somewhat different approach here. For a selected set of 13 genes that had been previously reported to be altered in this same brain region in 57 SCZ cases relative to 57 matched controls among the Pitt cohort (Supplementary Fig. 3), we compared results from RNA-seq to that of qPCR when these quantifications are taken from different tissue samples, although they were taken from the same subject and roughly the same brain region. Therefore our results also account for possible differences in pathological sampling of brain region and variability in RNA extraction.
