Gene-specific primers were used in PCR reactions (20µl) containing 7µl of ddH2O, 10µl of 2xSYBR Green MasterMix (Bio-Rad), 1µl of each specific primer (10mM), and 1µl of first-strand cDNA template. The qPCR program included an initial denaturation for 3 min at 95°C followed by 40 cycles of denaturation at 95°C for 10s, annealing for 30s at 55°C, and extension for 30s at 72°C. For melting curve analysis, a dissociation step cycle (55°C for 10s, and then 0.5°C for 10s until 95°C) was added. All primers were tested for efficiency prior to use, and only primer pairs with the same efficiency as the primers for housekeeping genes were used. Primers for 18S and 28S were obtained from Pan et al. (Pan et al. , 2015). Candidate gene-specific primers used were as follows (F, forward primer; R, reverse primer): DPOGS201379-F , 5’- CTGACCAGCACGAAGAGAAA-3’;DPOGS201379-R , 5’- GACAATATCCCGGCGAATAGAA-3’;DPOGS211203-F , 5’-GATGCGATTGCTGCATTGAATA-3’;DPOGS211203-R , 5’- ATACCGCTGCCATCACTAAC-3’; DPOGS202675 -F, 5’-CTCCCTTGTCGTGATGTTGT-3’; DPOGS202675 -R, 5’-GTCGGCTCTCAATCCAGTAAA -3’; DPOGS200868 -F, 5’-TCGGAACAGGAGGAGTATCT-3’; DPOGS200868 -R, 5’-GCCTCTATGCCTCTCTTCTATG-3’; DPOGS215054 -F, 5’-GTCGCTGACTTCTCCATCATAC-3’; DPOGS215054 -R; 5’-GTTCTCGTTCAGAGGATCCATATT-3’; DPOGS211604 -F, 5’-CAACGAGGAAGCCAGACTAAA-3’; DPOGS211604 -R, 5’-TGTGGCATTGGTCTTCCATAA-3’.
Due to homogeneity-of-variance and error normality assumptions, we could not use linear models to analyze gene expression. Instead, we used one-tailed Mann-Whitney U tests in R 3.1.3 (R Development Core Team, 2012) to determine whether gene expression was higher in eastern than western monarchs.