Transcriptomic analyses may be validated using an independent technique, for example, quantitative PCR (qPCR), which is recognisable and statistically assessable. Gene expression is measured against defined standards both for the gene of interest and control genes. The measurement by qPCR is similar to that obtained by RNA-Seq wherein a value can be calculated for the concentration of a target region in a given sample. qPCR is, however, restricted to amplicons smaller than 300 bp, usually toward the 3’ end of the coding region, avoiding the 3’UTR. If validation of transcript isoforms is required, an inspection of RNA-Seq read alignments should indicate where qPCR primers might be placed for maximum discrimination. The measurement of multiple control genes along with the genes of interest produces a stable reference within a biological context. qPCR validation of RNA-Seq data has generally shown that different RNA-Seq methods are highly correlated.
Functional validation of key genes is an important consideration for post transcriptome planning. Observed gene expression patterns may be functionally linked to a phenotype by an independent knock-down/rescue study in the organism of interest.