Stably Expressed Housekeeping Genes across Developmental Stages in the Two-Spotted Spider Mite, Tetranychus urticae

Abstract

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring mRNA expression. To facilitate gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to stable housekeeping genes is mandatory. In this study, ten housekeeping genes, including beta-actin (Actin) , elongation factor 1 α (EF1A) , glyceralde hyde-3-phosphate dehydrogenase (GAPDH) , ribosomal protein L13 (RPL13) , ribosomal protein 49 (RP49) , α-tubulin (Tubulin) , vacuolar-type H+-ATPase (v-ATPase) , succinate dehydrogenase subunit A (SDHA) , 28S ribosomal RNA (28S) , and 18S ribosomal RNA (18S) from the two-spotted spider mite, Tetranychus urticae, were selected as the candidate reference genes. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the performance of these candidates as endogenous controls across different developmental stages. In addition, RefFinder, which integrates the above-mentioned software tools, provided the overall ranking of the stability/suitability of these candidate reference genes. Among them, PRL13 and v-ATPase were the two most stable housekeeping genes across different developmental stages. This work is the first step toward establishing a standardized qRT-PCR analysis in T. urticae following the MIQE guideline. With the recent release of the T. urticae genome, results from this study provide a critical piece for the subsequent genomics and functional genomics research in this emerging model system.

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus

“…challenged intestine, gill and hepatic caecum with LPS). Average Ct value from three biological replicates was further analyzed according to previous studies55. The stability of thirteen RGs was analyzed by five algorithms: geNorm28, NormFinder29, BestKeeper30, deltaCt method31 and RefFinder32.…”

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

“…Due to its high accuracy, sensitivity, specificity, and rapidity, RT-qPCR has been widely used for validating the results of gene expression profiles [35]. However, RT-qPCR requires normalization by reference genes to offset variation among samples processed from different tissues [36]. In recent years, with the gradual deepening of insect molecular biology research, it is becoming increasingly necessary to select appropriate reference genes to evaluate many biological processes or important functional genes in insects [2,3,22,24,29,[37][38][39][40][41][42][43][44][45][46][47].…”

Selection of Reference Genes for RT-qPCR Analysis Under Extrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis

“…Consequently, the selection of appropriate reference genes is critical in offsetting variations and biases (Lü et al, 2018). Recently, there has been a dramatic increase in studies of reference gene selection in the cell-content feeding arthropods, including Hemiptera ( Supplementary Table S3; Li et al, 2013;Yang et al, 2014Yang et al, , 2015bYuan et al, 2014;Shang et al, 2015;Koramutla et al, 2016;Ma et al, 2016;An et al, 2016;Yu et al, 2016;Arya et al, 2017;Kang et al, 2017), and three spider mites species (Sun et al, 2010;Niu et al, 2012;Yue et al, 2013;Yang et al, 2015a). Most of these studies in Tetranychidae species have focused on the expression patterns of reference genes under the impact of insecticide and developmental stage.…”

“…Recently, selection of reference genes has been conducted for an array of cell-content feeding arthropods, including three spider mites species, Tetranychus urticae (Yang et al, 2015a), Tetranychus cinnabarinu (Sun et al, 2010), and Panonychus citri (Niu et al, 2012), and many Hemiptera species, Myzus persicae (Kang et al, 2017), Aphis gossypii (Ma et al, 2016), Toxoptera citricida (Shang et al, 2015), Lipaphis erysimi (Koramutla et al, 2016), Sogatella furcifera , Aphis craccivora (Yang et al, 2015b), Bemisia tabaci (Li et al, 2013), Phenacoccus solenopsis (Arya et al, 2017), Nilaparvata lugens (Yuan et al, 2014), Acyrthosiphon pisum (Yang et al, 2014), and Ericerus pela (Yu et al, 2016). In addition, we previously screened for the internal references under intrinsic conditions from a pool of A. viennensis HKGs (Yang et al, 2019).…”