Abstract
Simple SummaryReference genes are critical for standardizing expression data of RT-qPCR across samples of organisms under different experimental conditions. However, most commonly used reference genes may not be stably expressed leading to a risk of misinterpretation of results. In our study, nine reference genes were evaluated in Tuta absoluta (a destructive pest of tomato) at different developmental stages, tissues, 20-hydroxyecdysone (20E) and insecticide treatments. Finally, the expression profile of indicator gene EcR after 20E treatment was evaluated to verify the accuracy of the results. This study is essential for improving accuracy and reliability to normalize gene expression data in T. absoluta and provides a useful strategy for other insects.The tomato leaf miner, Tuta absoluta is a destructive pest of tomato. The leaf-mining activities of its larvae can cause significant yield losses. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used to measure gene expression, and the selection of stable reference genes for calibration and standardization is critical for accurate use of RT-qPCR. We studied the stable expression of nine common housekeeping genes in T. absoluta. These were examined at different developmental stages, in larval tissues, as well as those induced by exposure to 20E and insecticides. Four dedicated algorithms (geNorm, BestKeeper, NormFinder, and ΔCt method) and online tool (RefFinder) were used to analyze and rank the tested reference genes. Based on the standardized gene expression data of target gene ecdysone receptor (EcR), the applicability of specific reference genes was verified. The results clarify that the optimal internal reference genes vary greatly under different experimental conditions. GAPDH and RPS11 were the best reference genes for developmental stages; RPL28 and RPL10 for different tissues; EF1α and RPL28 for 20E treatment; EF1α and RPL7A for insecticide treatments. The most suitable reference genes in all experimental conditions are EF1α and RPL28.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for nucleic acid quantification due to its sensitivity, specificity, and reproducibility [1]
The RT-qPCR efficiency ranged from 92.9% (RPL28) to 109.1% (SOD), and regression coefficient ranged from 0.991 (ACT) to 0.998 (AK and ribosomal protein L10 (RPL10)) (Table 2)
The results indicated that all primers met the standard requirement for RT-qPCR analyses [6]
Summary
Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for nucleic acid quantification due to its sensitivity, specificity, and reproducibility [1]. Stable reference genes are necessary for normalization [4]. Elongation factor 1-α (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin (ACT) are constitutively expressed genes that are commonly used as reference genes [8,9]. It is unclear if the expression of these genes are always stable. The exact experimental conditions for the expression of each candidate reference gene must be verified to avoid the ambiguity of data in RT-qPCR analysis [11]
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