Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata  

作　　者：濮菲 杨丙晔 柯才焕 

机构地区：[1]State Key Laboratory of Marine Environmental Science, Xiamen University [2]College of Ocean and Earth Science, Xiamen University [3]Xiamen Medical College

出　　处：《Chinese Journal of Oceanology and Limnology》2015年第4期838-845,共8页中国海洋湖沼学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China(No.41176113);the National Basic Research Program of China(973 Program)(No.2010CB126403);the Changjiang Scholars Program for Innovative Research Team in Universities(No.IRT0941);the Earmarked Fund for Modern Agro-Industry Technology Research System(No.nycytx-47)

摘　　要：Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 （ACT-2）, elongation factor 1 alpha （EF-1α）, elongation factor 1 beta （EF-1β）, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, ubiquitin （UBQ）, β-tubulin （β-TUB）, and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene （Gβ） across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.Accurate quantification of transcripts using quantitative real-time polymerase chain reaction(q PCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2(A CT- 2), elongation factor 1 alpha(E F- 1α), elongation factor 1 beta(E F- 1β), glyceraldehyde-3-phosphate dehydrogenase(G APDH), ubiquitin( UBQ), β-tubulin(β- TUB), and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using q PCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the ge Norm software to assess the expression stability of the candidate reference genes, revealing that β- TUB and UBQ were the most stable genes. The commonly used G APDH and 18 SrRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene( Gβ) across tissue types was also examined and normalized to the expression of each or both of U BQ and β- TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β- TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in C rassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

关 键 词：Crassostrea angulata gene expression quantitative real-time PCR internal control gene G protein β-subunit gene 

分 类 号：S917.4[农业科学—水产科学]