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.