实时荧光定量PCR的三种数据分析方法比较

摘要

目的 比较实时荧光定量PCR的三种数据分析方法(2-△△CT、REST2009 和REST MCS) 的异同.方法 以正常小鼠来源的cDNA 混合物阳性对照为模板,5 倍系列稀释,分别做miRNA-181a 和Sn RNA U6 的标准曲线.实时荧光定量PCR检测正常组和哮喘组小鼠脾脏CD4+T 细胞miRNA-181a和Sn RNA U6 表达水平,以Sn RNA U6 为内参基因,采用2-△△CT、REST2009 和REST MCS 方法,对正常组和哮喘组miRNA-181a 进行相对定量分析.结果 5倍系列稀释法制作的标准曲线提示miRNA-181a 和Sn RNA U6 基因扩增效率分别为99.8%和99.9%,采用2-△△CT提示,REST2009 软件分析,RESTMCS 软件分析,皆表明哮喘组miRNA-181a 的表达量为正常组的2.92 倍,但是三种方法在适用条件、分析数据的多少等方面存在一定的区别.结论 三种数据分析方法各有特点,可根据研究者本人的实验要求选择合适的方法.%Objective To compare three data analysis methods for relative quantification in real-time quantitative PCR (i.e. 2-ΔΔCT, REST2009 and REST MCS). Methods Using 5-fold serial dilutions of positive control cDNA mixture as template, the standard curves of miRNA -181a and Sn RNA U6 were created, respectively. The expression levels of miRNA-181a and Sn RNA U6 in the CD4+T lymphocyte between the normal control group and the asthma group were measured by qRT-PCR, and the relative quantification of miRNA-181a between the two groups was analyzed by three methods of 2-ΔΔCT, REST2009 and REST MCS, respectively. Sn RNA U6 was used to normalize miRNA-181a qRT-PCR data. Results The amplification efficiencies of miRNA-181a and Sn RNA U6 were 99.8% and 99.9% respectively. Calculated by 2-ΔΔCT, REST 2009, and REST MCS,the expression level of miRNA-181a in the spleen CD4+ T cells in the asthma group was all 2.92 times as great as in the normal control group (P<0.05) . On the other hand, there were some differences in the indications and limitations of three methods. Conclusion These three methods are all useful in relative quantification, and the right method can be chosen according to the test requirement.