Validation of microarray data with qPCR

Gene expression data from the microarray measurement of TCN1 were validated with quantitative real-time PCR (qPCR). From the discovery sample, 94 individuals representing all diagnostic categories and spanning the full range of microarray expression levels were picked out for validation. High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation) was used for reverse transcription of 1 µg RNA. The qPCR reaction was performed on 5 ng cDNA using TCN1 TaqMan Gene Expression Assay (Hs01055542_m1; Life Technologies Corporation) as target. All samples were run in four replicates. 12 individuals were excluded due to poor cDNA quality. The results were analyzed with qbase+ software (Biogazelle, Zwijnaarde, Belgium) using the comparative cycling threshold (ΔΔCt) method⁷⁴ with GUSB (Hs00939627_m1) as endogenous control and a positive cDNA control sample as reference. The qPCR measures of TCN1 expression were regressed against the log2 microarray TCN1 gene expression values using a linear model in R 3.4.1.