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4 October 2018 Selection of Reference Genes for Optimal Normalization of Quantitative Real-Time Polymerase Chain Reaction Results for Diaphorina citri Adults
Shuying Bin, Xinhua Pu, Benshui Shu, Cong Kang, Shaoming Luo, Yu Tang, Zhongzhen Wu, Jintian Lin
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Abstract

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), can cause direct damage to citrus trees and is the main vector for the devastating disease, citrus greening disease or huanglongbing. Most molecular studies on this important insect pest use real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to quantify gene expression, including analyzing molecular basis for insecticide resistance in field populations. One critical factor to cause inaccuracy in RT-qPCR results is the lack of appropriate internal reference genes for optimal data normalization. In this study, the expression levels of 10 selected reference genes were evaluated in different tissue samples of psyllid adults and in the insects treated with different temperatures and insecticides. Data were analyzed using different computational algorithms, including Delta Ct, BestKeeper, NormFinder, geNorm, and RefFinder. According to our results, at least two reference genes should be used for the normalization of RT-qPCR data in this insect. The best choices of reference genes for different samples are as follows: ACT1 and Ferritin for different tissue samples, RPS20 and Ferritin for samples treated with different temperatures, TBP and EF1α for samples treated with imidacloprid, and Ferritin and TBP for samples treated with beta-cypermethrin. The reference genes identified in this study should be useful for future studies to analyze the expression patterns of target genes, especially for genes linked with temperature adaptability and insecticide resistance in this insect species in the future.

© Crown copyright 2018.© Crown copyright 2018.
Shuying Bin, Xinhua Pu, Benshui Shu, Cong Kang, Shaoming Luo, Yu Tang, Zhongzhen Wu, and Jintian Lin "Selection of Reference Genes for Optimal Normalization of Quantitative Real-Time Polymerase Chain Reaction Results for Diaphorina citri Adults," Journal of Economic Entomology 112(1), 355-363, (4 October 2018). https://doi.org/10.1093/jee/toy297
Received: 4 May 2018; Accepted: 6 September 2018; Published: 4 October 2018
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KEYWORDS
Diaphorina citri
real-time reverse-transcription quantitative polymerase chain reaction
Reference gene
stability evaluation
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