RNAi in the red flour beetle (Tribolium)

Abstract

Tribolium castaneum is exceptionally amenable to gene knockdown by RNA interference (RNAi) which, in this insect, is systemic (spreading throughout the organism and to the next generation), highly penetrant, and able to phenocopy genetic null phenotypes. Hence, any gene function can be knocked down at any stage in (apparently) all tissues upon injection of double-stranded RNA (dsRNA). The RNAi effect is elicited both in the injected animal and, if female pupae or adults have been injected, transferred to the offspring. Embryonic RNAi (eRNAi) usually generates the strongest phenotypes in the injected individual, but suffers from elevated lethality caused by injection injury. Pupal RNAi (pRNAi), in which female pupae are injected and phenotypes scored in the offspring, is the easiest to perform. However, in some cases, the knockdown of a gene leads to sterility of the injected female. This problem can be circumvented in many cases by injecting adult females (aRNAi) or using eRNAi. In order to interfere with processes during metamorphosis, injection into last-stage larvae is used (lRNAi). Up to two genes in a single experiment have been successfully knocked down via RNAi. The inclusion of more than two genes usually leads to a dilution effect, which lowers phenotypic strength. This protocol describes the production of dsRNA from a polymerase chain reaction (PCR) template, injection procedures for each Tribolium life stage, and important controls for effective analysis.