Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

PloS One
Yongcheng WeiLiuyi Xu

Abstract

Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected referen...Continue Reading

References

Nov 22, 2000·Journal of Biochemical and Biophysical Methods·T D Schmittgen, B A Zakrajsek
Jun 23, 2004·Journal of Experimental Botany·Claire GachonBénédicte Charrier
Jun 16, 2005·Journal of Molecular Endocrinology·S A BustinM W Pfaffl
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Feb 23, 2010·Plant Physiology and Biochemistry : PPB·Cecilia Vásquez-RobinetRuth Grene

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Citations

Jun 22, 2017·International Journal of Molecular Sciences·Yu LiuBingru Huang
Jul 13, 2018·The Science of the Total Environment·Mario SoliñoJosé M García Del Barrio

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Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

Primer3
geNorm
Excel
BestKeeper
NormFinder

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