Identification of suitable endogenous control genes for microRNA gene expression analysis in Pacific oyster

【作者】 黄雯； 阙华勇； 张国范； Fei Xu； Wei Wang； Li Li；

【Author】 Wen Huang;Fei Xu;Wei Wang;Li Li;Huayong Que;Guofan Zhang;Institute of Oceanology,Chinese Academy of Sciences;University of Chinese Academy of Sciences;

【机构】 中国科学院海洋研究所； Institute of Oceanology,Chinese Academy of Sciences；

【摘要】 We have previously conducted deep sequencing experiments on Pacific oyster(Crassostrea gigas)and have acquired 68 microRNAs(miRNAs) together with their expression pattern.To validate the expression profile and determine the function of these miRNAs,a real-time quantitative PCR(qRT-PCR) approach was commonly used,which relies on the reliable and stably expressed endogenous control genes for normalization.In this study,we identified suitable endogenous control genes in C.gigas for miRNA gene expression analysis.We compared the performance of5.8S r RNA,snRNA U1,snRNA U6,and several stably expressed miRNAs.Results suggested that the combination of miR-71 and miR-8 was the optimum choice for stably expressed endogenous control across the assayed development stages,whereas the combination of let-7 and miR-87 was the optimum choice for different organs.With the newly identified endogenous control genes,we validated the expression patterns of two randomly selected miRNA genes in organ samples and three miRNA genes in developmental samples.We also corrected the dubious expression profile of miR-100,let-7,and lin-4/miR-125 at early development stages.We validated as well the robust up regulated expression in pediveligers,which suggested that the three miRNAs may be involved in the settlement and metamorphosis of Pacific oyster.更多还原

【Abstract】 We have previously conducted deep sequencing experiments on Pacific oyster(Crassostrea gigas)and have acquired 68 microRNAs(miRNAs) together with their expression pattern.To validate the expression profile and determine the function of these miRNAs,a real-time quantitative PCR(qRT-PCR) approach was commonly used,which relies on the reliable and stably expressed endogenous control genes for normalization.In this study,we identified suitable endogenous control genes in C.gigas for miRNA gene expression analysis.We compared the performance of5.8S r RNA,snRNA U1,snRNA U6,and several stably expressed miRNAs.Results suggested that the combination of miR-71 and miR-8 was the optimum choice for stably expressed endogenous control across the assayed development stages,whereas the combination of let-7 and miR-87 was the optimum choice for different organs.With the newly identified endogenous control genes,we validated the expression patterns of two randomly selected miRNA genes in organ samples and three miRNA genes in developmental samples.We also corrected the dubious expression profile of miR-100,let-7,and lin-4/miR-125 at early development stages.We validated as well the robust up regulated expression in pediveligers,which suggested that the three miRNAs may be involved in the settlement and metamorphosis of Pacific oyster.更多还原