Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in <i>Sesamia inferens</i> (Lepidoptera: Noctuidae)

<div><p>The pink stem borer, <i>Sesamia inferens</i>, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in <i>S. inferens</i>. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (<i>18S rRNA</i>), elongation factor 1 (<i>EF1</i>), glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>), ribosomal protein S13 (<i>RPS13</i>), ribosomal protein S20 (<i>RPS20</i>), tubulin (<i>TUB</i>), and β-actin (<i>ACTB</i>) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (<i>RPS13</i>, <i>RPS20</i>, and <i>EF1</i>) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). <i>18S rRNA</i>, <i>EF1</i>, and <i>GAPDH</i> were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). <i>18S rRNA</i>, <i>RPS20</i>, and <i>TUB</i> were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (<i>hsp83</i>) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in <i>S. inferens</i>.</p>