qPCR validation of RNAseq data

To validate RNAseq findings, brains from a separate cohort of HAZ and LAZ animals exposed to MIA for 1h but not used for RNAseq were processed for qPCR (n = 6/group). As described above, RNA was extracted using peqGOLD Trifast (Thermo Scientific) followed by DNase digestion (Ambion DNA-free™ DNA Removal Kit, Thermo Scientific) and then reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to manufacturer instructions. For relative quantification of mRNA levels, qPCR was performed on a CFX384 Touch™ Real-Time PCR Detection System (BioRad, Vienna, Austria) using SsoAdvanced Universal SYBR Green Supermix (Biorad) [18]. Six protein-coding genes (as3mt, neuropeptide Y receptor Y8b, cd164, ptgr1, frizzled class receptor 4 and collagen, type XII, alpha 1b) were semi-randomly selected across levels of significance, fold change and direction of expression change during the RNAseq experiment and primers for these genes were designed with Primer BLAST (Additional file 1: Table 11). Correct sequence amplification was confirmed by Sanger sequencing. All samples were measured as triplicates. actin beta1 and ribosomal protein L13a, which were not differentially expressed in the RNAseq dataset, were used as reference genes for quantification of target gene expression. Quantitative measurements of target gene levels relative to LAZm were performed with the ΔΔ Cq method using the mean value of the LAZm group as the calibrator. Group differences were expressed as fold changes and then converted to log2 fold change to enable comparison with the RNAseq dataset.