Abstract
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. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin, GAPDH, RPL13, RPL6, RPL32, RPS18, and ATPB, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the ΔCt method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1, 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 H. vigintioctopunctata.
Highlights
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a frequently used technique for gene expression studies on account of its high specificity, high sensitivity, high throughput, and low cost (Hellemans and Vandesompele, 2014)
All of these candidate reference genes were expressed in H. vigintioctopunctata and intuitional with a single amplicon of the expected size for each gene (Supplementary Figure S1)
Five papers have been published for the reference gene selection of the whitefly, Bemisia tabaci, a notorious and invasive insect species, in the past 5 years (Li et al, 2013; Su et al, 2013; Collins et al, 2014; Liang et al, 2014; Dai et al, 2017)
Summary
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a frequently used technique for gene expression studies on account of its high specificity, high sensitivity, high throughput, and low cost (Hellemans and Vandesompele, 2014). RT-qPCR involves standardization to the expression of a battery of appropriately stable reference genes concurrent. H. vigintioctopunctata colonizes many different species of plants, for example, solanaceous plants such as eggplant, tomato, potato, and pepper; cucurbitaceous plants such as cucumber, white gourd, and loofah. It attacks many weeds, such as the black nightshade, winter cherry, thorn apple, and tobacco (Pang and Mao, 1979). The destructive potential of H. vigintioctopunctata is high at both the adult and larval stages, leading to up to 60% loss of fruit production (Sharma et al, 2012; Kawazu, 2014)
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