RNA-seq validation of RNAi identifies additional gene connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae)

Authors: Perkin, Lindsey; Oppert, Brenda; Gerken, Alison

Submitted to: Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/9/2017
Publication Date: 4/21/2017
Publication URL: https://handle.nal.usda.gov/10113/5801887
Citation: Perkin, L.C., Oppert, B.S., Gerken, A.R. 2017. RNA-seq validation of RNAi identifies additional gene connectivity in Tribolium castaneum (Coleoptera: Tenebrionidae). Journal of Insect Science. 17(2):57;1-7. doi:10.1093/jisesa/iex026.

Interpretive Summary: RNA interference (RNAi) is a genetic tool to validate phenotypes and gene function by reducing gene expression. Traditionally, after RNAi, reduced gene expression is checked using quantitative polymerase chain reaction (qPCR). However, this technique is limited in that it only checks one gene at a time. We propose to use RNA sequencing (RNAseq) to check for changes in gene expression across all genes in the genome. We demonstrate this technique using two genes, a common control gene and a gene that does not show a visual change after RNAi. We found that using RNAseq, we were able to show a reduction in target gene expression as well as changes in many other genes, some unexpected. This new way of quantifying gene expression after RNAi will improve the way we monitor gene expression changes and elucidate new gene function and gene connections.

Technical Abstract: RNA interference (RNAi) is a functional genomics tool to validate phenotypes by delivering targeted, gene-specific, and complementary dsRNA into a host via injection, feeding, or other means in order to reduce gene expression. RNAi in the red flour beetle, Tribolium castaneum, has been successful due to a response to injected dsRNA at any life stage. Traditionally, successful transcript knockdown has been quantified by qPCR on a gene-by-gene basis, where only expression of the target gene and normalization genes are evaluated. In this study, we provide two examples of gene-specific knockdown via injected RNAi, but instead of qPCR, we use RNA-seq to quantify gene expression of the transcriptome. We show that this method can give insight into the “complete” transcriptome response associated with a phenotype, while still providing validation of the target knockdown. The first example explores the knockdown of a commonly used positive phenotypic control gene, aspartate 1-decarboxylase (ADC), which gives a reliable phenotype of an adult with a black cuticle instead of the wild-type red-brown. The second gene, chitin deacetylase 6 (CDA6), a chitin-modifying enzyme localized to the T. castaneum anterior and posterior midgut, does not show a visible phenotype following injection of dsRNA. We find in both cases that target gene expression is reduced, but also demonstrate that other genes are significantly differentially expressed, providing clues to the possibility of interconnected metabolic pathways.