论文部分内容阅读
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。
A suitable internal reference gene is a prerequisite for accurate analysis of relative gene expression using qRT-PCR. In order to accurately and quantitatively analyze differentially expressed genes between antler growth stem cells and antler stem cells, TATA box binding protein (TBP), actin beta (ACTB), microtubules Tubulin alpha 1a (TUBA1A), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) The 6 genes of β2-microglobulin (B2M) were used as internal reference genes. The qRT-PCR technique was used to detect the expression of m RNA in antler growth center and antler stem cells respectively. A comprehensive assessment. The results of Delta-Ct method, ge Norm (ver.3.5), Bestkeeper (ver.1.0) and NormFinder (ver.0.953) and Ref Finder software showed that the expression of ACTB and TUBA1A genes were the highest among antler stem cells. B2M and ACTB genes expressed the highest stability among antler growth center cells, and B2M and ACTB genes showed the highest stability among all deer cell lines. Therefore, B2M, ACTB and TUBA1A candidate genes can be used as internal control genes for differential expression of antler growth stem cells and antler stem cells. This study provides a theoretical basis for further research on antler growth and development and application of antler models in biomedical research .