Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae)
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Abstract
Quantitative real-time PCR (qRT-PCR) is a powerful technique for measuring and evaluating gene expressions during different biological processes. To facilitate gene expression studies, normalization with respect to stable housekeeping genes (HKGs) is mandatory. The western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), the main vector of Tomato spotted wilt virus (TSWV), is a very destructive invasive species. In this study, expression profiles of 11 candidate HKGs, including β-actin (Actin), α-tubulin (Tubulin), elongation factor 1 α (EF1A), vacuolar-typeH+-ATPase (ATPase), NADH-ubiquinone oxidoreductase (NADH), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), ribosomal protein l32 (RPL32), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S), from no nviruliferous and viruliferous F. occidentalis were investigated. Four distinct algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to determine the performance of these genes as endogenous controls under the virus condition. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidates, HSP70 , HSP60, EF1A, and RPL32 were the most stable housekeeping genes. This work is the initial first step to establish a standardized qRT-PCR analysis in F. occidentalis. Additionally, this study lays a foundation for the research in the interactions between TSWV and F. occidentalis.
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2014. Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae) PeerJ PrePrints 2:e662v1 https://doi.org/10.7287/peerj.preprints.662v1Author comment
This is a submission to PeerJ for review.
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Supplemental Information
The mean and standard deviation (SD) of the Ct value for each candidate reference gene
The mean and standard deviation (SD) of the Ct value for each candidate reference gene .
Summary of mean and SD values of gene pairwise comparison using the ΔCt method
Ranking of the candidate reference genes by BestKeeper
The agrose gel electrophoresis of the eleven candidate reference genes
The agrose gel electrophoresis of the eleven candidate reference genes. M, DL 2000 bp Marker; Templates in the PCR reactions were as follows: 1) 18S; 2) 28S; 3) Actin; 4) ATPase; 5) EF1A; 6) HSP60; 7) HSP70; 8) HSP90; 9) NADH; 10) RPL32; 11) Tubulin.
Melting curves of the eleven candidate reference genes
Melting curves of the eleven candidate reference genes
Additional Information
Competing Interests
The authors declare they have no competing interests.
Author Contributions
Chunxiao Chunxiao Yang conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Hui Li performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Huipeng Pan conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Yabin Ma performed the experiments, reviewed drafts of the paper.
Deyong Zhang contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Yong Liu contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Zhanhong Zhang contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Changying Zheng contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Dong Chu conceived and designed the experiments, wrote the paper, reviewed drafts of the paper.
Funding
This research was supported by a Special Fund for Agroscience Research in the Public Interest (Award Agreement No.: 201303028), the Science and Technology Development Planning Program of Qingdao (13-1-3-108-nsh), and the Shandong Modern Agricultural Technology & Industry System (SDAIT-02-021-11). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
