摘要
目的建立特异、稳定且可靠的血浆microRNA（miRNAs）实时荧光定量PCR技术。方法收集10例健康人血浆标本,采用mirVanaTM PARIS kit试剂盒法提取血浆miRNAs,以miRNA特异的茎环引物引导逆转录,通过SYBR Green实时荧光定量聚合酶链反应（PCR）对外参照cel-miR-39、cel-miR-238及血浆miR-342-3p进行检测。结果健康人血浆miR-342-3p及外参cel-miR-39、cel-miR-238可以实现特异性扩增及定量。溶解曲线显示10例健康人血浆标本中cel-miR-39、cel-miR-238和miR-342-3p扩增产物分别在81.44、81.62、82.71℃时出现单一峰,无引物二聚体峰和其他非特异峰出现。血浆miR-342-3p批内批间重复性实验的标准差在0.13～0.20、变异系数（CV）在0.42%～0.66%,显示该方法重复性较好。以cel-miR-39、cel-miR-238作为外参照,同一血浆标本miR-342-3p的5次检测结果ΔCt的标准差为0.22、CV为1.68%,说明cel-miR0-39、cel-miR-238可作为血浆miRNAs实时荧光定量PCR检测的稳定外参。结论实时荧光定量PCR技术可作为研究血浆miRNAs较好的技术平台。
Objective To establish a specific, stable and reliable real-time fluorescence quantitative PCR for detecting plasma microRNAs（miRNAs）. Methods The plasma samples from 10 healthy individuals were collected,and miRNAs was extracted using mirVanaTM PARIS kit. Exogenous cel-miR-39 and cel-miR-238 and endogenous plasma miRNAs were reversely translated by specific stem-loop primers and quantified by real time fluorescence quantitative PCR. Results cel-miR-39,cel-miR-238 and miR-342-3p were amplified and quantified specifically in RNA preparations isolated from plasma samples of healthy individuals. The amplification products of cel-miR-39, cel-miR-238 and miR-342-3p showed a single melting peak at 81.44,81.62 and 82.71℃, respectively, without primer dimer peak or non-specific peak in all 10 cases of healthy individual plasma samples. The standard deviation（SD） of intra-assay and extra-assay of miR-342-3p was 0.13-0. 20, and the coefficient of variation（CV） was 0.42%- 0.66 %, which suggesting that this detection method has a good repeatability. The levels of miR-342-3p were detected in a same plasma sample, each experiment was repeated for 5 times,and normalized by cel-miR-39 and eel-miR-238. The SD and CV of ACt was 0.22,1. 68% ,respectively,which indicating that cel-miR-39 and cel-miR-238 could be taken as the stable exogenous reference for the plasma miR- NAs detection by real-time fluorescence quantitative PCR. Conclusion Real-time fluorescence quantitative PCR could serve as a good platform for plasma microRNA research.
出处
《国际检验医学杂志》
CAS
2015年第1期57-59,共3页
International Journal of Laboratory Medicine
