Abstract
Empoasca onukii Matsuda is one of the most devastating pests of the tea plant (Camellia sinensis). Still, the presumed expression stability of its reference genes (RGs) has not been analyzed. RGs are essential for accurate and reliable gene expression analysis, so this absence has hampered the study of the insect’s molecular biology. To find candidate RGs for normalizing gene expression data, we cloned ten common housekeeping genes from E. onukii. Using the ΔCt method, geNorm, NormFinder and BestKeeper, we screened the RGs that were appropriate for quantifying the mRNA transcription of cellular responses under five experimental conditions. We identified the combinations of α-TUB and G6PDH, α-TUB and UBC, two RGs (α-TUB and β-TUB1) or three RGs (α-TUB, RPL13 and GAPDH), AK and UBC, or RPL13 and α-TUB as the best for analyzing gene expression in E. onukii adults of both sexes in different tissues, nymphs at different developmental stages, nymphs exposed to different temperatures or nymphs exposed to photoperiod stress. Finally, the E. onukii cysteine proteinase (Eocyp) was chosen as the target gene to validate the rationality of the proposed RGs. In conclusion, our study suggests a series of RGs with which to study the gene expression profiles of E. onukii that have been manipulated (biotically or abiotically) using reverse transcription quantitative polymerase chain reaction. The results offer a solid foundation for further studies of the molecular biology of E. onukii.
Highlights
Reverse transcription quantitative polymerase chain reaction (RT-qPCR, hereafter qPCR) is a popular and indispensable technique for quantifying the mRNA transcription of cellular responses triggered by biotic or abiotic manipulations [1]. qPCR offers high-throughput screening, and is known to be fast, sensitive and accurate [2,3,4]
Our results identify a series of reference genes (RGs) that could be used with qPCR to study the gene expression profiles of E. onukii treated with induced stresses; these will provide a solid foundation for further studies of the molecular biology of E. onukii
Little attention has been paid to the presumed expression stability of RGs and to gene expression analysis in E. onukii
Summary
Reverse transcription quantitative polymerase chain reaction (RT-qPCR, hereafter qPCR) is a popular and indispensable technique for quantifying the mRNA transcription of cellular responses triggered by biotic or abiotic manipulations [1]. qPCR offers high-throughput screening, and is known to be fast, sensitive and accurate [2,3,4]. Every step of qPCR sample preparation and processing—determining the intrinsic variability of RNA, removing impurities during RNA extraction, carrying out reverse transcription and measuring PCR efficiencies —needs to be accurately normalized [5,6,7]. Reference genes for Empoasca onukii are the best internal controls when results are quantified using the 2-ΔΔCt method or its modified versions [8, 9]. The measurement of internal controls along with target genes helps to compensate for the inevitable experimental variations, such as disparities in the amount of starting material and/or sample loading [10, 11]. Identifying appropriate RGs for a normalization scalar is an essential prerequisite for developing a qPCR assay. At least two RGs (preferably more) should be employed simultaneously in the normalization process [12,13,14,15]
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