title: Selection of reference genes for quantitative real-time RT-PCR analysis in citrus.
abstract: Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR.
authors.affiliation: Horticulture and Landscape College, Hunan Agricultural University, Furong District, Changsha, 410128 Hunan, China. yanjw200709@126.com
authors.forename: Jiawen
authors.initials: J
authors.lastname: Yan
authors.forename: Feirong
authors.initials: F
authors.lastname: Yuan
authors.forename: Guiyou
authors.initials: G
authors.lastname: Long
authors.forename: Lei
authors.initials: L
authors.lastname: Qin
authors.forename: Ziniu
authors.initials: Z
authors.lastname: Deng
mesh_terms: D002957:Citrus / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S