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目的筛选出在北柴胡不同生长时期及不同器官中表达均稳定的内参基因并进行验证。方法利用实时荧光定量PCR获得18个候选内参基因所有Ct值,通过3种不同算法(Bestkeeper、Norm Finder、Ge Norm)的软件对内参基因稳定性进行分析,采用皮尔森相关系数(Pearson correlation coefficient)分析3个软件给出的稳定性排名结果。结果所有候选内参基因的Ct值相对宽泛,ADF1b、ADF5、ADF7、e IF2b和ACT2为最为合适的内参基因,而e IF6被认为稳定性最差的内参基因。3个软件计算结果均呈现显著性相关。结论采用实时荧光定量PCR结合3种不同算法进行北柴胡内参基因的筛选及验证是可行的。在北柴胡柴胡分子遗传研究中,发掘到的内参基因对目的基因进行均一化处理有助于提高目的基因表达分析的精确性及可信度。
Objective To screen and validate the internal reference genes that are stable in different growth stages and organs of Bupleurum chinense. Methods All Ct values of 18 candidate reference genes were obtained by real-time fluorescence quantitative PCR. The stability of the reference genes was analyzed by softwares of three different algorithms (Bestkeeper, Norm Finder, Ge Norm). The Pearson correlation coefficient Analysis of 3 software gives the stability ranking results. Results The Ct value of all candidate internal reference genes was relatively broad. ADF1b, ADF5, ADF7, e IF2b and ACT2 were the most suitable internal control genes, while e IF6 was the least stable internal control gene. The results of three softwares showed significant correlation. Conclusion The real-time fluorescence quantitative PCR combined with three different algorithms for screening and validation of Bupleurum internal reference gene is feasible. In the study of Bupleurum chinensis molecular genetic research, the discovery of the reference gene to homogenize the target gene will help to improve the accuracy and reliability of the gene expression analysis.