RT-qPCR Validation of Candidate Genes Underlying Thermal Adaptation in Drosophila subobscura

Understanding the genetic basis of thermal adaptation is essential in the face of global climate change. In this study, we validate candidate genes implicated in thermal adaptation previously identified through RNA-seq analysis of Drosophila subobscura populations experimentally evolved under a progressive warming regime. Using RT-qPCR, we assessed the directionality of gene expression changes - up- or downregulation -across warming and control populations from two latitudinal origins. Out of 27 candidate gene tests, 33.3% showed consistent expression patterns between RNA-seq and RT-qPCR. Our results suggest that effect size, rather than expression level alone, is a key factor driving successful validation. This should be considered when interpreting whole-transcriptome data, which can yield many candidate genes. Future studies should examine how different log2 fold-change thresholds between populations relate to the success rate of validating RNA-seq differential expression results, in order to improve the reliability of candidate gene lists.