The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected fiv...The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.展开更多
基金Supported by the funding for school-level research projects of Yancheng Institute of Technology(No.xjr2019047)the National Natural Science Foundation of China(No.31902362)。
文摘The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.
