实时荧光定量PCR的三种数据分析方法比较.doc

窑128窑 热带病与寄生虫学 圆园12 年第 10 卷第 3 期 允燥怎则灶葬造 燥枣 栽则燥责蚤糟葬造 阅蚤泽藻葬泽藻泽 葬灶凿 孕葬则葬泽蚤贼燥造燥早赠 圆园12援 V燥l 10. 晕燥 3 doi:10.3969/j.issn.1672-2302.2012.03.002 实时荧光定量 PCR 的三种数据分析方法比较 酆孟洁 邱晨 * 刘雯雯 揖摘要铱 目的 比较实时荧光定量 PCR 的三种数据分析方法 (2原吟吟CT尧REST2009 和 REST MCS) 的异同遥 方法 以正常小鼠来源的 cDNA 混合物阳性对照为模板袁5 倍系列稀释袁分别做 miRNA-181a 和Sn RNA U6 的标准曲线遥 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 表达水平袁以 Sn RNA U6 为内参基因袁采用 2原吟吟CT尧REST2009 和 REST MCS 方法袁对正常组和哮喘组 miRNA-181a 进行相对定量分析遥 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%袁 采用 2原吟吟CT 提示袁REST2009 软件分析袁REST MCS 软件分析袁皆表明哮喘组 miRNA-181a 的表达量为正常组的 2.92 倍袁但是三种方法在适用条件尧分析数据的多少等方面存在一定的区别遥 结论 三种数据分析方法各有特点袁 可根据研究者本人的实验要求选择合适的方法遥 揖关键词铱 实时荧光定量聚合酶链式反应曰 相对定量曰 标准曲线曰 微小 RNA Comparison of three data analysis methods for relative quantification in real-time quantitative PCR Feng Mengjie, Qiu Chen*, Liu W enwen. Department of Respiratory Diseases, Second Clinical Medical Col鄄lege of Jinan University袁Shenzhen People爷s Hospital袁Shenzhen袁518020袁China. *Corresponding author. 揖Abstract铱 Objective To compare three data analysis methods for relative quantification in real -time quantitative PCR 渊i.e. 2原吟吟CT, REST2009 and REST MCS冤. Methods Using 5-fold serial dilutions of posi鄄 tive 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 原 吟吟CT, REST2009 and REST MCS, respectively. Sn RNA U6 was used to normalize miRNA-181a qRT-PCR data. Results The ampli鄄 fication efficiencies of miRNA-181a and Sn RNA U6 were 99.8% and 99.9% respectively. Calculated by 2原吟吟CT, 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 grou