A practical guide for functional transcriptomics: a case study in RNA interference and qPCR to understand the explosive chemistry of Brachinus bombardier beetles (Coleoptera: Carabidae).

Abstract Transcriptomics stands to benefit functional studies by providing a direct path to developing genome-wide toolboxes in almost any insect system. This chapter aims to communicate the best practices in insect functional transcriptomics, defined as the use of transcriptomic data to identify and validate genes that express phenotypes of interest. A workflow is described for identifying candidate genes based on de novo transcriptome assemblies, functionally validating candidates using RNA interference (RNAi), and quantifying knockdown expression levels with quantitative PCR (qPCR). The chapter emphasizes 'universal' steps that could apply to any transcriptome study once an assembled, annotated transcriptome is obtained. These include design and delivery of short interfering RNA (siRNA) used for RNAi, identification of qPCR reference genes, and design of qPCR primers. As a case study, results are presented of an investigation into the chemical defense system of the bombardier beetle Brachinus elongatulus.