改良通用茎环引物筛查和定量检测微小RNA方法的建立

The application of universal stem loop primer for microRNA scanning and quantification

目的 建立可同时筛选和定量检测成熟型miR的荧光定量PCR方法.方法 改良通用茎环引物,在其茎环尾端引入8个随机碱基片段,用于成熟型miR逆转录,以建立SYBR Green PCR筛选和定量检测miR的方法（简称“改良通用茎环引物miR法”）.同时,采用10倍梯度稀释的miR-155标准品cDNA（1～109拷贝/μl）评价其灵敏度；采用熔解曲线评价其检测miR-155的特异性；通过对2×105、2×106、2×107拷贝/μl的miR-155标准品cDNA分别进行批内20次重复实验,以其循环阈值（Ct）的变异系数（CV）评价其精密度；并与传统茎环法进行比较,计算实验所用时间和引物成本.然后,采用改良通用茎环引物miR法对植物血凝素（PHA）刺激培养0、16、24、48、72 h的人外周血T淋巴细胞内miR进行筛选（87个靶miR,经PubMed检索可能与免疫功能相关）,并用以进行miR定量检测分析.结果 建立的改良通用茎环引物法的最低检测限为103拷贝/μl的miR-155 cDNA标准品；熔解曲线于80℃呈单峰,证实为针对miR-155的特异性扩增；其批内重复实验的精密度为CV＜2.5％.优化后的通用引物方法与传统方法相比较,节约引物约75％（1 917 bp比7 851 bp）,节省逆转录时间约120min（85 min比205 min）.用改良通用茎环引物法筛选T淋巴细胞中87个miR,85个miR在PHA刺激前、后的Ct无变化,而miR-150和miR-155表达量在T淋巴细胞激活前后分别发生超过10倍下降（72 h）和8倍的升高（48 h）,且差异有统计学意义（Z=-2.023,P=0.043;Z=-2.032,P=0.042）.结论 建立的改良通用茎环引物miR法具有快速、重复性好、灵敏、节约引物用量和实验时间的优点,可用于T淋巴细胞的miR筛查和定量检测.

Objective To establish a universal stem loop primer （USLP） based real-time PCR method to scan mature miR profile and quantify it＇s expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ＇ end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR （improved USLP method）.10-fold gradient diluted standard miRNA-155 cDNA （ 1 ～ 109 copies/μ1） were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation （ICV） of threshold cycle （Ct value） through 20 repeated detections of the standard miR-155 cDNA （2 × 105,2 × 106,2 × 107 copies/μl） ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin （PHA） for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable （ICV 〈 2.5% ）.Compared with the traditional stem loop primers,our method saved 75% cost of primers （ 1 917 bp vs 7 851 bp） and 60% test time of reverse transcription （85 min vs 205 min）.By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 （72 h） decreased by 10 times and that of miR-155 （48 h） increased 8 times after culture with PHA （Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ）.Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.