茎环引物实时定量PCR技术用于检测异核苷修饰的siRNA的可行性研究

Feasibility of quantification for isoNA modified siRNA using stem-loop RT-qPCR

本文考察了茎环引物实时定量PCR技术用于定量检测D-/L-异核苷(D-/L-iso NA)修饰的siRNA的可行性.根据阈值循环数(Ct值)及异核苷修饰的siRNA模板的初始浓度的对数值(lgC)绘制了标准曲线,与siRNA的标准曲线进行对比,发现D-异尿苷(D-isodU)修饰的siRNA(D-isodU-siRNA)不影响茎环引物实时定量PCR过程,而L-isodU修饰的siRNA(L-isodU-siRNA)降低了逆转录效率,而使测得的Ct值偏高,且对逆转录效率的影响有浓度依赖性和修饰位点特异性.因此,对前者既可进行绝对定量也可进行相对定量,对于后者只能进行绝对定量.将上述方法应用于isodU-siRNA血清稳定性的检测,获得了更可靠的定量结果.运用双标准曲线法,可进行胞内isodU-siRNA的定量检测.

In this work, the feasibility of quantification for isoNA modified siRNA using stem-loop RT-qPCR was researched. The standard curve was drawn with the cycle threshold（Ct） and the logarithm of initial siRNA concentration（lgC）. Comparing with natural siRNA, the incorporation of D-isod U into siRNA had no influence on stem-loop RT-qPCR, while the incorporation of L-isod U reduced the reverse transcription efficiency and resulted in higher Ct value. The decrease of amplification efficiency caused by L-isod U varied with the initial siRNA concentration and modified position. Thus, absolute quantification could be used in both D-isod U and L-isod U modified siRNAs, while relative quantitation could only be used in D-isod U-siRNAs. Stem-loop RT-qPCR could be used in the qualitative analysis of isod U-siRNA serum stability. Besides, the dual-standard curve could also be used to quantify the intracellular isod U-siRNA.