Selection of reference genes for gene expression studies in Siberian Apricot (Prunus sibirica L.) Germplasm using quantitative real-time PCR

Jun Niu; Baoqing Zhu; Jian Cai; Peixue Li; Libing Wang; Huitang Dai; Lin Qiu; Haiyan Yu; Denglong Ha; Haiyan Zhao; Zhixiang Zhang; Shanzhi Lin

doi:10.1371/journal.pone.0103900

Selection of reference genes for gene expression studies in Siberian Apricot (Prunus sibirica L.) Germplasm using quantitative real-time PCR

PLoS One. 2014 Aug 8;9(8):e103900. doi: 10.1371/journal.pone.0103900. eCollection 2014.

Authors

Jun Niu¹, Baoqing Zhu¹, Jian Cai¹, Peixue Li², Libing Wang³, Huitang Dai², Lin Qiu², Haiyan Yu³, Denglong Ha², Haiyan Zhao², Zhixiang Zhang¹, Shanzhi Lin¹

Affiliations

¹ College of Biological Sciences and Biotechnology, College of Nature Conservation, National Engineering Laboratory for Tree Breeding, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing Forestry University, Beijing, China.
² Jigongshan National Nature Reserve, Xingyang, China.
³ Research Institute of Forestry, Chinese Academy of Forestry, Beijing, China.

Abstract

Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ) in nine Siberian Apricot Germplasms (including much variability) were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

MeSH terms

Computational Biology
DNA Primers / genetics
DNA, Complementary / genetics
Gene Expression Profiling / methods*
Gene Expression Regulation, Plant / genetics*
Genes, Plant / genetics*
Prunus / genetics*
Prunus / metabolism
Real-Time Polymerase Chain Reaction / methods
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction / methods

Substances

DNA Primers
DNA, Complementary

Grants and funding

This research was supported by the National Natural Sciences Foundation of China (No. J1103516), the Central Public-Interest Scientific Institution Basal Research Fund (RIF2013-02) (No. 201004001) and the Chinese Key Technology Research and Development Program of Twelfth Five-Year Plan (No. 2011BAD22B08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.