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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 compare the differences of micro RNA (miRNA) between plasma and stem cells by poly-A polymerase (PAP) and real-time quantitative polymerase chain reaction (PCR) , And evaluated the stability of expression of two commonly used internal control genes. Methods The mi RNAs were extracted from plasma of adult Sprague Dawley rats. The expression of rno-mi R-200b-3p and rno-mi R-126-3p in the plasma was detected by stem-loop and PAP plus real-time quantitative PCR. mi R-103a-3p and U6 were used as internal controls, and the two ΔCt methods were used to compare the two real-time quantitation methods and the choice of internal reference. Results The stem-loop method could reduce the Ct value by 2 to 4 cycles compared with the PAP plus tail method, and the detection sensitivity increased ten-fold. The peak of the dissolution curve of PAP plus tail showed a small peak before obvious single peak, and the stem-loop method showed an independent single peak , Stem-loop specificity is better; U6 as internal reference relative to rno-mi R-103a more stable. Conclusions Stem-loop method is accurate, sensitive and specific for the detection of a small amount of mi RNA. For the detection of large amount of mi RNA and large-scale tissue samples, the PAP tailing rule is more suitable. For the selection of internal reference genes, semi-quantitative miRNA using U6 is more stable and reproducible than rno-mi R-103a-3p.