两种实时定量聚合酶链反应方法检测血浆微量微RNA的比较

Detection of plasmatic trace amounts of microRNAs with two kinds of small RNA realtime polymerase chain reaction methods

目的比较用茎环和多腺苷酸聚合酶(poly A polymerase,PAP)加尾实时定量聚合酶链反应(polymerase chain reaction,PCR)法检测血浆中微量微RNA(micro RNA,mi RNA)的区别,并评价2种常用内参基因的表达稳定性。方法分离成年Sprague Dawley大鼠血浆提取mi RNA,使用茎环和PAP加尾实时定量PCR法检测血浆中rno-mi R-200b-3p和rno-mi R-126-3p的表达水平,采用rno-mi R-103a-3p和U6作为内参,采用2△Ct法计算比较两种实时定量方法和内参的选择。结果茎环法相较于PAP加尾法检测Ct值可以降低2~4个循环数,检测灵敏度增加了10倍;PAP加尾法溶解曲线在明显单峰之前可见小峰,茎环法显示独立单峰,茎环法特异性更优;U6作为内参相对于rno-mi R-103a更稳定。结论对于mi RNA浓度偏低的少量样本检测,茎环法有准确、灵敏、特异的优点;对于mi RNA含量丰富、大规模组织样本的检测,PAP加尾法则更适用。对于内参基因的选择,使用U6进行mi RNA半定量相对于rno-mi R-103a-3p更稳定,重复性更强。

Objective To detect traces of microRNAs （miRNAs） in plasma and assess the expression stability of two common reference genes by stem-loop and poly A polymerase （PAP） real-time quantitative polymerase chain reaction （PCR） method, as miRNAs are the new bio-markers of tumor diagnosis and molecular targeted therapy, and its quantitative research is very important. Methods We extracted miRNAs from plasma of adult Sprague Dawley （SD） rats＇ plasma, and detected the expressions of rno-miR-200b-3p and rno-miR-126-3p with stem-loop and PAP real-time PCR quantitative method, with rno-miR-103a-3p and U6 as internal controls. All the results were evaluated by 2^△Ct method. Results Compared with PAP method, the stem-loop method reduced Ct value by 2-4 cycles and improved sensitivity by 10 times. In PAP method, the melting curve showed two peaks, a main peak and a small non-specific peak. Yet the melting curve of stem-loop method demonstrated a single specific peak. Furthermore, we validated the stability of internal references in the two real time PCR methods. U6 presented a more stable Ct value than rno-miR-103 in adult SD rats＇ plasma samples. Conclusions Stem-loop real-time PCR is recommended as a major way to detect some samples with a low concentration of miRNAs, owing to its high accuracy and sensitivity. However, if a large number of tissue samples is going to be detected, PAP real-time PCR is more suitable and convenient than stern-loop method. U6 is more stable and repeatable than rno-miR-103a-3p as the reference gene to evaluate the semi-quantitative consequence of miRNAs.