Identification of stable reference genes for real-time quantitative PCR (RT-qPCR) in Locusta migratoria infected by Paranosema locustae
Author of the article:WEI Xiao-Jia;HE Lan;ADILA·Aji;JI Rong;HU Hong-Xia
Author's Workplace:International Center for the Collaborative Management of Cross-border Pest in Central Asia, College of Life Sciences, Xinjiang Normal University, Urumqi 830054, China
Key Words:Paranosema locustae; Locusta migratoria; real-time PCR; reference genes; expression stability
Abstract:
[Objectives] To identify stable reference genes in Locusta migratoria infected by paranosema locustae for real-time quantitative PCR (RT-qPCR). [Methods] In this study, five commonly used candidate L. migratoria reference genes (glyceralde-hyde-3-phosphate dehydrogenase,GAPDH. 18sRNA. tubulin,TUB. actin,ACT. And elongation factor1,EF1) were evaluated to assess their suitability for use in the normalization of RT-qPCR data in locusts infected with different concentrations of Paranosema locustae spores(1×104、1×106 and 1×108 spores/individual). Gene expression profiles were analyzed using the geNorm, Normfinder, and BestKeeper software packages. [Results] geNorm predicted TUB to be the most, and 18S the least, stable reference genes based on their respective M-values (0.2784 and 0.6584). NormFinder estimates of the stability values (SV) of each candidate reference gene were EF1(0.152), ACT(0.181), TUB(0.212), GAPDH(0.329) and 18S(0.395), which allows these genes to be ranked in order of stability as follows; EF1 > ACT > TUB > GAPDH > 18S. BestKeeper evaluates the expression stability of reference genes by calculating and comparing their variance, including their coefficient of variance (CV) and standard deviation (SD). This analysis indicated that all five genes were stable on the basis of having SD values < 1.0. However, with respect to CV values, the ACT gene was the most stable. [Conclusion] These results indicate that the best reference genes for L. migratoria infected with P. locustae are ACT、EF1 and TUB.