Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae)

Simple Summary Quantitative real-time fluorescent polymerase chain reaction (qRT-PCR) is a momentous tool for calculating the expression levels of targeted genes across various experimental conditions. The selection and evaluation of stable reference genes for qRT-PCR analysis is an essential precondition for reliable expression assessment. Phthorimaea operculella is one of the most serious Lepidopteran pests that attack potatoes around the world. In the present paper, a total of 10 commonly used reference genes, namely ACT, alpha-TUB, 18S, 28S, GAPDH, EF1 alpha, RPL4, RPL13, RPL27 and SOD, were selected and validated for suitability under three treatments (developmental stages, tissues/organs and temperatures) using five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). These results indicated that EF1 alpha and RPL13 were the best suitable reference genes for diverse backgrounds. The relative transcript levels of the target gene chitin synthase A gene (PoChSA) were abundantly expressed in epidermal cells, and lowly transcribed in the midgut. Our findings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in P. operculella. Due to a lack of effective internal references, studies on functional genes in Phthorimaea operculella, a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (ACT), alpha-tubulin (alpha-TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1 alpha), 18S and 28S ribosomal RNA (18S, 28S), ribosomal protein genes RPL4, RPL13 and RPL27 and superoxide dismutase (SOD) were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that RPL13, EF1 alpha and RPL27 are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; EF1 alpha and RPL13 are recommended to normalize the gene expression levels among diverse tissues. EF1 alpha and RPL13 are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, EF1 alpha and RPL13, we estimated the tissue-biased expression level of chitin synthase A gene (PoChSA). As expected, PoChSA was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of P. operculella.

Automated summary

Based on quantitative real-time fluorescent polymerase chain reaction (qRT-PCR) analysis, we selected and validated 10 commonly used reference genes and identified EF1 alpha and RPL13 as the most suitable reference genes for different experimental conditions. Our findings will contribute to improving the accuracy of qRT-PCR analysis of target gene expression in P. operculella.