Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.
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This work was supported by the Special Fund for Agroscience Research in the Public Interest (201303028), the Shandong Modern Agricultural Technology & Industry System (SDAIT-02-021-11), the Science and Technology Development Planning Program of Qingdao (13-1-3-108-nsh), and the Taishan Mountain Scholar Constructive Engineering Foundation of Shandong, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Yang, Chunxiao; Li, Hui; Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; and Chu, Dong, "Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis" (2015). Entomology Faculty Publications. 91.
S1 Fig. The agrose gel electrophoresis of the eleven candidate reference genes.
journal.pone.0135207.s002.TIF (1669 kB)
S2 Fig. Melting curves of eleven candidate reference genes in <em>Frankliniella occidentalis</em>.
journal.pone.0135207.s003.DOCX (11 kB)
S1 Table. The mean and standard deviation (SD) of the <em>C<sub>t</sub></em> value for each candidate reference gene.
journal.pone.0135207.s004.DOCX (14 kB)
S2 Table. Summary of mean and SD values of gene pairwise comparison using the <em>ΔC<sub>t</sub></em> method.