Validation of RNA-Seq data by quantitative PCR

To validate RNA-Seq data, the transcript levels of 15 genes (Table 2) were examined by quantitative reverse transcriptase PCR (qRT-PCR). Genes were chosen by their notable differential expression between feeding states in male beetles. Aliquots consisting of approximately 500 ng of total RNA from a subset of samples (fed male replicate 4, unfed male replicate 4, fed female replicate 4, unfed female replicate 4, fed male replicate 1, unfed male replicate 3, unfed female replicate 2) were used to make cDNA using iScript™ Reverse Transcription Supermix (Bio Rad, Hercules, CA). PCR was conducted in a 20 μL reaction consisting of iTaq™ Universal SYBR® Green Supermix (Bio Rad) and 2 μL of template for 40 cycles of 95 °C for 5 s and 60 °C for 30 s on a Bio Rad CFX96 Real-Time PCR Machine (Bio Rad). Primers designed to amplify specific transcripts of these genes were designed using IDT Primer Quest and melt curves were produced to ensure primer specificity and proper PCR temperature cycling parameters (Additional file 1). For each cDNA sample the PCR reactions were conducted in triplicate and relative target gene expression was normalized to that of YQE_05788, which encodes ribosomal protein S3P. Ribosomal protein S3 is a relatively more stable normalizing gene for qRT-PCR in another beetle, Tribolium castaneum, compared to the more routinely used actin or tubulin genes [29]. Fold change was calculated for each normalized gene in relation to the expression of the unfed male treatment using the 2^-ΔΔCT method [30]. For each gene, Pearson and Spearman Correlation Coefficients were computed between the seven samples measured by qRT-PCR and RNA-Seq.

Correlation coefficients of RNA-Seq and qRT-PCR measured expression levels across seven samples and 15 genes