AUTHOR=Shen Chen-Hui , Tang Min , Li Xiao-Fei , Zhu Li , Li Wei , Deng Pan , Zhai Qing , Wu Gang , Yan Xiao-Hong 

TITLE=Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae)

JOURNAL=Frontiers in Physiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2024.1345836

DOI=10.3389/fphys.2024.1345836

ISSN=1664-042X

ABSTRACT=<p><italic>Mylabris sibirica</italic> is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in <italic>Mylabris sibirica</italic>, have been tremendously limited. In this study, ten internal reference genes (<italic>ACT</italic>, <italic>ARF1</italic>, <italic>AK</italic>, <italic>EF1α</italic>, <italic>GAPDH</italic>, <italic>α-TUB</italic>, <italic>RPL6</italic>, <italic>RPL13</italic>, <italic>RPS3</italic> and <italic>RPS18</italic>) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that <italic>RPL6</italic> and <italic>RPL13</italic> were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of <italic>RPL6</italic> and <italic>RPS18</italic> was recommended to test gene transcription levels under different adult tissues. <italic>AK</italic> and <italic>RPL6</italic> were the best reference genes in male and female adults. <italic>RPL6</italic> and <italic>RPL13</italic> were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (<italic>MsUAP</italic>), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in <italic>M. sibirica</italic>, such as development, reproduction, sex differentiation, cold and heat resistance.</p>