title: Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.
abstract: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.
authors.affiliation: National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Gao
authors.affiliation: National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Ma
authors.affiliation: National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Shuai
authors.affiliation: National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Junyi
authors.initials: J
authors.lastname: Gai
authors.affiliation: National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000535:Aluminum / Q000378:metabolism*; D002104:Cadmium / Q000378:metabolism*; D002681:China; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006358:Hot Temperature; D010940:Plant Proteins / Q000235:genetics; D018517:Plant Roots / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013025:Glycine max / Q000235:genetics* / Q000378:metabolism; D013312:Stress, Physiological / Q000235:genetics*
chemical_list: D010940:Plant Proteins; D002104:Cadmium; D000535:Aluminum