Screening and validation of reference genes using in RT-qPCR for gene expression studies in Paederus fuscipes, a medically and agriculturally important insect

Paederus fuscipes is a medically and agriculturally important insect all over the world. P. fuscipes cause not only a skin condition but is also an aggressive predator in different agro-ecosystems. Prior to performing RT-qPCR under a variety of conditions to investigate the function of target genes in P. fuscipes, it is essential to screen reference genes. However, no P. fuscipes reference gene(s) has yet been discovered. Using the RefFinder software package, which includes Delta Ct, geNorm, NormFinder, and BestKeeper, we evaluated the stability of seven housekeeping genes of P. fuscipes under five conditions (developmental stage, diet, temperature, tissue and sex). During the RT-qPCR analysis, geNorm software was used to evaluate pairwise variation in order to identify the most appropriate number of reference genes. The results revealed that for developmental stages RPL-13, EF1A and beta-tubulin; for sex-related experiments beta-tubulin and Actin, for tissue-related experiments PRS-18, 18S and RPL-13; for temperature-related experiments beta-tubulin and PRL-13 and diet-related experiments, beta-tubulin and PRS-18 are suitable reference genes. Furthermore, the associated HSP-70 (as the reporter gene) expression patterns differed significantly when the three most stable and three least stable reference genes were selected in different temperature treatments. This is the first time that a complete list of standardized reference genes has been given for P. fuscipes to use in an RT-qPCR study, which could be helpful for potential functional studies of target genes in P. fuscipes. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of King Saud University.