Total RNA was extracted from ∼90 heads or 30 bodies using TRIzol (Ambion, Life Technologies). RNA samples were treated with DNase (Ambion DNA-Free Kit), and equal amounts of RNA (1 μg) were reverse-transcribed into complementary DNA (Applied Biosystems). Biological and technical replicates were then analyzed with SYBR Green Real-Time PCR (ABI PRISM 7700 Sequence Detection System; Bio-Rad) and performed using the following PCR conditions: 39 cycles (30 s at 95°, 30 s at 60°, 30 s at 72°). The Ct threshold was manually adjusted to 0.6 across all samples and targets. Then, each of the target genes was normalized to rp49 expression for the comparative ΔCt method analysis following subsequent comparison to the control genotype to assess fold enrichment (ΔΔCT method). Primers, listed below, were all tested for efficiency before using for experiments.
