Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae)

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1 Hunan Academy of Agricultural Sciences, Institute of Plant Protection, Changsha, Hunan, China
2 Qingdao Agricultural University, College of Agronomy and Plant Protection, Qingdao, Shandong, China
3 University of Kentucky, Department of Entomology, Lexington, Kentucky, United States
4 Hunan Vegetable Institute, Hunan Academy of Agricultural Sciences, Changsha, Hunan, China
DOI
10.7287/peerj.preprints.662v1
Published
Accepted
Subject Areas
Entomology, Genomics, Molecular Biology, Virology
Keywords
Frankliniella occidentalis, reference genes, qRT-PCR analysis, Tomato spotted wilt virus
Copyright
© 2014 Yang et al.
Licence
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
Cite this article
Yang CC, Li H, Pan H, Ma Y, Zhang D, Liu Y, Zhang Z, Zheng C, Chu D. 2014. Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae) PeerJ PrePrints 2:e662v1

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.

Author Comment

This is a submission to PeerJ for review.

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 .

DOI: 10.7287/peerj.preprints.662v1/supp-1

Summary of mean and SD values of gene pairwise comparison using the ΔCt method

DOI: 10.7287/peerj.preprints.662v1/supp-2

Ranking of the candidate reference genes by BestKeeper

DOI: 10.7287/peerj.preprints.662v1/supp-3

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.

DOI: 10.7287/peerj.preprints.662v1/supp-4

Melting curves of the eleven candidate reference genes

Melting curves of the eleven candidate reference genes

DOI: 10.7287/peerj.preprints.662v1/supp-5