OpenArray high-throughput qPCR
TaqMan® OpenArray® chips from Applied Biosystems (Burlington, ON, Canada) were used to quantify transcription at the 54 genes (50 candidate and 4 endogenous control genes) on a QuantStudio 12K Flex Real‐Time PCR System following the manufacturer’s protocol. Forty-eight cDNA samples were run (two chips for 48 samples) for each of the 54 genes on each chip. A 5 μL reaction volume which includes 1.2 μL of cDNA (100ng/µL/per sample), 1.3 μL of ddH2O and 2.5 μL of TaqMan® OpenArray® Real‐Time PCR Master Mix (Applied Biosystems, Burlington, ON, Canada) was used, aliquoted across a 384‐well plate and then loaded onto the TaqMan® OpenArray® chips using the OpenArray® AccuFill System. A total of 10 chips were used for 213 cDNA samples. The samples were randomly distributed among the chips. ExpressionSuite Software (Applied Biosystems, Thermo Fisher Scientific, Carlsbad, CA, USA) was used to analyse the endogenous control genes. Of four endogenous control genes, β-Actin was selected for normalization due to lower among-sample variation compared to the three other endogenous control genes. Subsequently, all 10 chips were normalized with the selected endogenous control gene (β-Actin) together in ExpressionSuite Software v1.0.3 (Applied Biosystems, Burlington, Ontario, Canada). Moreover, ExpressionSuite Software was used to calculate raw critical threshold (CT) values and the relative critical threshold values (ΔCT). Values produced by this platform are already corrected for the efficiency of the amplification (Molina-Lopez et al., 2020). We tested for replicate effect using Paired sample T test in SPSS (IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp). As we found no evidence for a replicate effect (P value > 0.05), CT and ΔCTvalues were averaged between the replicate and only one CT or ΔCT value was used for each gene.