利用qRT-PCR技术筛选鹿茸生长中心细胞与鹿茸干细胞内参基因

发布时间：2018-03-10 01:05

  本文选题：鹿茸　切入点：干细胞　出处：《农业生物技术学报》2017年05期 　论文类型：期刊论文

【摘要】：合适的内参基因是利用qRT-PCR准确分析基因相对表达量的先决条件。为了对鹿茸生长中心细胞和鹿茸干细胞间的差异表达基因进行准确定量分析,本研究筛选了TATA盒结合蛋白(TATA box binding protein,TBP)、β-肌动蛋白(actin beta,ACTB)、微管蛋白1α(tubulin alpha 1a,TUBA1A)、葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PDH)、3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)及β2-微球蛋白(β2-microglobulin,B2M)这6个基因作为内参基因,利用qRT-PCR技术分别检测这些基因m RNA在鹿茸生长中心细胞及鹿茸干细胞中的表达情况,并对其表达的稳定性进行了综合评估。运用Delta-Ct method,ge Norm(ver.3.5)、Bestkeeper(ver.1.0)和NormFinder(ver.0.953)和Ref Finder软件综合分析表明,在鹿茸干细胞之间,ACTB和TUBA1A基因表达稳定性最高;在鹿茸生长中心细胞之间,B2M和ACTB基因表达稳定性最高;在所有鹿细胞系之间,B2M和ACTB基因表达稳定性最高。因此,B2M、ACTB以及TUBA1A候选基因可作为研究鹿茸生长中心细胞及鹿茸干细胞基因差异表达的内参基因,本研究为进一步研究鹿茸生长发育机制和开展鹿茸模型应用于生物医学研究提供了一定的理论依据。
[Abstract]:In order to analyze the differentially expressed genes between antler growth center cells and pilose antler stem cells accurately, it is a prerequisite for accurate analysis of relative expression by qRT-PCR. In this study, Tata box binding TATA cassette binding protein, 尾 -actin betaan ACTB, tubulin 1 伪 tubulin alpha TUBA1A, glucose-6-phosphate dehydrogenase G6PDH3-phosphate glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 尾 2-microglobulinB2M (尾 2-microglobulinB2M) were selected as internal reference genes. QRT-PCR technique was used to detect the expression of m RNA in antler growth center cells and pilose antler stem cells, and the stability of the expression was evaluated. The results of Delta-Ct methodge Normver.3.5Bestkeeperver.1.0) and Norm Finderver.0.953) and Ref Finder software showed that, The stability of ACTB and TUBA1A gene expression was the highest in the pilose antler stem cells, and the stability of B2M and ACTB gene expression was the highest in the pilose antler growth center cells. The stability of B2M and ACTB gene expression was the highest among all deer cell lines. Therefore, the candidate genes of B2M ACTB and TUBA1A could be used as internal reference genes to study the differentially expressed genes of pilose antler growth center cells and pilose antler stem cells. This study provides a theoretical basis for further study on the growth and development mechanism of velvet antler and the application of antler model in biomedical research.
【作者单位】： 中国农业科学院特产研究所特种经济动物分子生物学国家重点实验室/吉林省鹿茸工程研究中心;
【基金】：国家自然科学基金(No.31500792和No.31402059) 中国农业科学院科技创新工程项目(No.CAAS-ASTIP-2014-ISAPS-04)
【分类号】：S825
，

本文编号：1591060