Evaluation of the expression stability of potential reference genes for RT-qPCR in Spodoptera frugipreda larvae exposed to camptothecin

Abstract

Camptothecin, a quinoline alkaloid, has strong action against Spodoptera frugiperda, a polyphagous pest found globally. Real-time quantitative PCR (RT-qPCR) is a popular and dependable technique for analyzing the mRNA expression of target genes. While RT-qPCR normalization requires reference genes with consistent expression. Many investigations have discovered that pesticides can change the expression patterns of reference genes. To date, the effects of CPT treatments on the expression stability of reference genes in S. frugiperda larvae are unclear. This study chose eight candidate reference genes, including alpha-tubulin (α-TUB), beta-1-tubulin (β-1-TUB), Actin, elongation factor 1 alpha (EF1α), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), and ribosomal protein L13 (RPL13). Five approaches were used to investigate expression stability in S. frugiperda larval samples treated with CPT: ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder, respectively. Furthermore, the ideal number of reference genes was determined using GeNorm. Our findings revealed that two reference genes were sufficient to normalize RT-qPCR in samples treated with CPT. The recommended reference gene combinations for different samples are as follows: α-TUB and β-1-TUB for CPT-treated larval samples; β-1-TUB and RPL13 for samples of larval cuticle tissues; RPL3 and RPL13 for the larval fat body samples; EF1α and RPL3 for the larval malpighian tube samples; and EF2 and Actin for the larval midgut samples. Our results laid the groundwork for the mRNA expression analysis of target genes in S. frugiperda impacted by CPT exposure, contributing to the research of the molecular action mechanism of CPT in S. frugiperda.