A total of 20 stage-enriched genes were selected for validation using qRT-PCR as described [29 (link)]. One microgram total RNA samples were reverse transcribed into first-strand cDNA using a SuperScript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) with oligo (dT) 15 primer. The cDNA products were diluted 20-fold with nuclease-free water before undertaking the qPCR. Each 25 μl PCR reaction contained 12.5 μl of 2 × Brilliant II SYBR Green QPCR Master Mix (Agilent, USA), 1 μl cDNA, 1 μl of the forward and reverse primer pair (Additional file 2 : Table S1), and 10.5 μl of sterile water. PCR cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 30 s denaturation at 95 °C and 1 min annealing and extension at 60 °C. A dissociation step (95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s and 60 °C for 15 s) was performed to confirm the amplification specificity for each gene. 26S proteasome non-ATPase regulatory subunit 4 (PSMD4) [29 (link), 40 (link)] was employed as a house-keeping gene in the assays. PCR reactions were performed in technical triplicates on the 7300 Real-Time PCR system (Applied Biosystems). The relative expression level of each gene was analysed using SDS 1.4 software (Applied Biosystems). Correlations between the microarray and qPCR results for 20 stage-enriched genes were analysed with the Spearman’s rho.
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