两种分析miRNA相对表达量方法的比较研究

目的基于实时反转录一聚合酶链反应（RT—PCR）基因表达相对定量的分析方法：GenEx软件分析与2（-△△CT）法，建立一种简便、准确的miRNA相对定量方法。方法以HK-2细胞系cDNA为模板，进行PCR扩增，确定最佳退火温度；再倍比稀释该模板，用荧光定量PCR测定的ct值作miR-122和miR-16的标准曲线，计算出相应miRNA的扩增效率。用荧光定量PCR测定健康者和乙型肝炎患者血清中内参miR-16和靶基因miR-122的表达，分别采用GenEx软件分析与2（-△△CT）法来评价相对表达量。结果系列稀释法制作的标准曲线呈现较好的线性相关性，两种miRNA的标准曲线r2值均为0．999，miR-16的扩增效率为97％，miR-122扩增效率为107％。2（-△△CT）法分析乙型肝炎患者血清中miR-122的表达较健康者增高了44．3倍，而GenEx软件分析为54．1倍。结论GenEx软件分析考虑了更多的影响因素，其结果比（-△△CT法更可靠，适用于临床分析。

Comparative study on two analysis methods of miRNA relative expression

Objective Based on the data obtained from the real-time RT-PCR,there are two methods for analy- sis： GenEx software analysis and 2（-△△CT） method. This study is to establish a simpIe and accurate microRNA relative quantitative method. Methods To determine the optimal annealing temperature,we amplified HK-2 cell line cDNA by PCR. Standard curve of miR-122 and miR-16 were made from the 10-fold diluted cDNA template from HK-2 cells. According to the curve,calculate respective miRNA amplification efficiency. The expression of target gene miR- 122 and reference gene miR-16 were determined by quantitative PCR. Base on the Ct data, the GenEx software analy- sis and 2〈-aacT） method were used to evaluate the miR-122 relative expression rate in the sera of healthy subjects and hepatitis B patients. Results A good linear correlation （r2 =0. 999） was obtained from two standard curve of miRNA genes. The amplification efficiency of miR-16 and miR-122 were 97 % and 107%, respectively. The expression of miR- 122 that 2（-△△CT） analysis received was 44.3 times higher in the serum of hepatitis B patients compared with healthy controls. But the rate that GenEx software analysis obtained was 54. 1. Conclusion Because the GenEx software a- nalysis considered more factors, so the result was more reliable and suitable for clinical application.