ABSTRACT Helopeltis antonii Signoret is an important sucking pest of cashew and other tree crops. Reverse transcription‐quantitative PCR (RT‐qPCR) is a widely used technique for analyzing gene expression. The selection of reliable reference genes is critical for accurate quantification of target gene expression using RT‐qPCR. There are no studies to identify reliable reference genes or gene expression studies in H. antonii . In this study, the expression stability of eight candidate genes, RPS3A, βTubulin1, RPL13A, 18S, EF1α, Ubiquitin‐conjugating enzyme E2 g (UBQ), GAPDH , and TBP , was evaluated across five different nymphal instars and different adult tissues and sexes of H. antonii . Results of stability analysis using multiple algorithms showed that the expression stability of candidate genes varied across different samples. Comprehensive stability ranking with RefFinder showed that RPL13A and TBP were the most stable in different tissues, RPS3A and UBQ were the most stable in different developmental stages, RPS3A and UBQ were the most stable in the two sexes, and UBQ and TUB were the most stable across all samples of H. antonii . Validation of identified stable reference genes ( UBQ and the combination of UBQ and TUB ) in normalization of two target genes ( COX1 and CSP13 ) expression provided consistent and biologically meaningful results. This study represents the first systematic study on the identification of reliable reference genes in H. antonii and provides the basis for future molecular investigations in this important pest species.
Savadi et al. (Thu,) studied this question.