2.4. Gene expression validation

To validate gene expression results from the RNA‐seq data, a subset of three differentially expressed transcripts involved in cuticular processes was evaluated through quantitative PCR (qPCR). Likewise, another three unannotated differentially expressed genes selected at random were also included in the qPCR analyses to confirm that no artifacts could be biasing gene expression patterns. Exon–intron boundaries were first identified by aligning the transcripts to wood tiger moth genomic data previously obtained through 454 pyrosequencing (Life Sciences) using Mummer v.3.23 (Kurtz et al., 2004). Bridging primers were then designed using Primer3 v. 4.0.0 (Untergasser et al., 2012). As a normalization controls (i.e., housekeeping genes), I selected one transcript (TRINITY_DN29483_c0_g2_i1) from the RNA‐seq data, which showed a uniform expression level within and between the two larval conditions, and a gene (GADPH) known to have stable expression in moth species in a variety of conditions (Lu et al., 2013). The software NormFinder v.5 (Andersen et al., 2004) was used to evaluate the normalized count matrix to find the transcript with the highest stability value and lowest expression variation within and between the two conditions. The annotation and primer sequences of the transcripts used for qPCR are presented in Table S1.

RNA for qPCR validation was extracted and purified as described above from the same caterpillars of both conditions. High‐quality RNA (200 ng) was converted into cDNA using iScript cDNA Synthesis Kit (Bio‐Rad). The specificity, dynamic range, and PCR efficiency of each primer pair were determined by testing against a 6‐step twofold dilution series of cDNA. All genes were amplified by triplicate (i.e., as technical replicates) in the three caterpillars from each condition (i.e., as biological replicates). All qPCRs (20 µl final volume) were run on a CFX96 (Bio‐Rad™) thermocycler using 300 nM of each primer, 10 µl iQ SYBR^® Green Supermix (Bio‐Rad™), and 4 µl of cDNA diluted fourfold. PCR conditions used throughout were 95°C for 3 min followed by 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 10 s. Melt curves were run after amplification to check for specificity from 55°C to 95°C with fluorescence readings taken in 0.5°C increments. Amplification efficiency of each gene was evaluated by plotting the standard curve Cq values against the log of the dilution factor for each point on the curve. The relative change in gene expression between the treatments was examined using the delta Ct method (Schmittgen & Livak, 2008).