实时定量PCR检测白血病相关miRNA方法的建立

本研究建立实时定量PCR（RQ-PCR）技术检测白血病相关miRNA的方法，探讨此方法在定量检测miRNA中的应用价值。通过提取82例慢性淋巴细胞白血病（CLL）、70例急性白血病（AL）患者骨髓或外周血标本总RNA，以cDNA为模板进行10倍梯度系列稀释，分别作miRNA及内参U6RNA的标准曲线并计算扩增效率；运用ABF7300定量PCR仪进行定量检测；采用SYBRGreen荧光染料法、以U6RNA作为内参照、循环阈值（ct）比较法进行miRNA表达相对定量分析，以检测CLL患者miR-15a、miR-16-1、miR-29b、miR-181b及AL患者miR．128．1、miR-223、let-7b、miR-155、miR-181a的表达。方法学考核参数包括特异性、线性范围、精密度和重复性。结果表明：RQ—PCR检测miRNA仅需10—50ng总RNA样本，检测下限为0．05ng，miRNA及u6的Ct值均在15—30之间，10倍系列稀释法制作的标准曲线呈现良好的线性关系（R^2〉0．980），扩增效率均在0．9以上，溶解曲线均为单峰，PCR产物经琼脂糖电泳均显示较亮的目的条带，批内差和批间差分别小于4．8％和6．3％。结论：RQ-PCR技术检测白血病相关miRNA敏感、快速，高通量，节约成本，定量范围宽，极大的方便了对miRNA的定量研究。

Detection of miRNA Levels in Leukemia Patients by Real-time Quantitative PCR

This study was purposed to establish a method for detecting miRNA expression by using fluorescent quantitative polymerase chain reaction （RQ-PCR）, and to investigate the application value of this method in quantitative detection of miRNA. Total RNA was extracted from the peripheral blood or bone marrow of 82 patients with chronic lymphocytic leukemia（CLL） and 70 patients with acute leukemias（AL）. Standard curves of U6 and miRNA were constructed from 10-fold serial dilutions of the cDNA, the quantitative detection was perfomed by using real-time quantitative PCR with SYBR Green by Roche Light Cycler. U6 RNA was used as the reference, the relative expression levels of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in patients with CLL, while miR-128-1, miR-223, let-7b, miR-155 and miR-181a in patients with AL were analyzed by 2（-△△CT） method. The relative parameters including specificity, linearity range, sensitivity and repeatability were evaluated. The results showed that the RQ-PCR assay could precisely detect the mature miRNA in as few as 10 - 50 ng of total RNA with the sensitivity of 0.05 ng RNA. Ct values of miRNA and U6 were all within 15 to 30. A good linear relationship （R^2 〉 0. 980） was obtained from the standard curves, also the efficiency of amplification was above 90%. Only a specific peak was shown on the melting curve diagram. The coefficients of variation （CV） of interrun and intrarun assay was 〈4.8% and 6.3% respectively. It is concluded that the RQ-PCR is a sensitive and fast method for the detection of miRNA with its high-flux and low cost, this method will facilitate the research with detection of miRNA.