AUTHOR=Lü Jing , Chen Shimin , Guo Mujuan , Ye Cuiyi , Qiu Baoli , Wu Jianhui , Yang Chunxiao , Pan Huipeng 

TITLE=Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctomaculata

JOURNAL=Frontiers in Physiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2018.01614

DOI=10.3389/fphys.2018.01614

ISSN=1664-042X

ABSTRACT=<p>Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. <italic>Henosepilachna vigintioctopunctata</italic> is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of <italic>H. vigintioctopunctata</italic> were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for <italic>H. vigintioctopunctata</italic> have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, <italic>Actin</italic>, <italic>GAPDH</italic>, <italic>RPL13</italic>, <italic>RPL6</italic>, <italic>RPL32</italic>, <italic>RPS18</italic>, and <italic>ATPB</italic>, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using <italic>RefFinder</italic>, which integrates four different analytical tools (<italic>Normfinder</italic>, <italic>geNorm</italic>, the Δ<italic>Ct</italic> method, and <italic>BestKeeper</italic>). The results displayed that <italic>RPL13</italic> and <italic>RPS18</italic> were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, <italic>lov</italic> and <italic>TBX1</italic>, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in <italic>H. vigintioctopunctata</italic>.</p>