CAJ | 학술논문

目的建立无需抽提RNA直接以血浆中的microRNAs (miRNAs)作为模板进行实时荧光定量PCR检测的新方法.方法方法学建立.采集健康人EDTA-K2抗凝静脉血并分离血浆,取7.5μl血浆用等量含2.5％(体积比)Tween20的缓冲液处理后进行逆转录及PCR,建立血浆miRNAs直接检测法.用该方法检测健康人血浆标本中U6 snRNA、miR-24-1-5p和miR-4634表达水平,评估其扩增的特异性和扩增效率.同时与传统的抽提RNA定量PCR检测法的阈值循环数(Cq)值进行比较并作相关性分析.另外,应用建立的血浆直接检测法,比较了混合引物和单个引物逆转录法检测健康人血浆标本中U6 snRNA、miR-24-1-5p和miR-4634结果的一致性.结果血浆直接检测法融解曲线显示健康人血浆标本中U6、miR-24-1-5p和miR-4634扩增产物分别在81、83、84.2 qC时出现单一峰,无引物二聚体峰和其他非特异峰出现.血浆直接检测法检测miR-24-1-5p扩增效率为1.1,同一份样本中U6、miR-24-1-5p和miR-4634这3种基因的扩增曲线平行,说明扩增斜率一致.直接检测法检测miR-4634的Cq值为30.98±0.17,高于RNA抽提法的29.22±0.21,差异有统计学意义(t=8.475,P＜0.01),而2种方法检测U6(直接检测法32.43±0.08,RNA抽提法32.57±0.06;t=1.354,P＞0.05)、miR-24-1-5p(直接检测法20.73±0.14,RNA抽提法21.06±0.21；t=1.749,P＞0.05)结果差异均无统计学意义.2种方法检测U6(r=0.860 5,P＜0.01)、miR-24-1-5p(r=0.728 9,P＜0.05)和miR-4634(r=0.748 5,P＜0.01)结果具有较好的相关性.混合引物和单个引物逆转录后PCR的Cq值之间无明显差异.结论建立的血浆直接检测法只需对血浆标本进行简单的预处理,并可使用混合引物进行逆转录定量PCR,具有操作简便、检测成本低等优点,值得进一步推广应用.
목적 건립무수추제RNA직접이혈장중적microRNAs (miRNAs)작위모판진행실시형광정량PCR검측적신방법.방법 방법학건립.채집건강인EDTA-K2항응정맥혈병분리혈장,취7.5μl혈장용등량함2.5％(체적비)Tween20적완충액처리후진행역전록급PCR,건립혈장miRNAs직접검측법.용해방법검측건강인혈장표본중U6 snRNA、miR-24-1-5p화miR-4634표체수평,평고기확증적특이성화확증효솔.동시여전통적추제RNA정량PCR검측법적역치순배수(Cq)치진행비교병작상관성분석.령외,응용건립적혈장직접검측법,비교료혼합인물화단개인물역전록법검측건강인혈장표본중U6 snRNA、miR-24-1-5p화miR-4634결과적일치성.결과 혈장직접검측법융해곡선현시건강인혈장표본중U6、miR-24-1-5p화miR-4634확증산물분별재81、83、84.2 qC시출현단일봉,무인물이취체봉화기타비특이봉출현.혈장직접검측법검측miR-24-1-5p확증효솔위1.1,동일빈양본중U6、miR-24-1-5p화miR-4634저3충기인적확증곡선평행,설명확증사솔일치.직접검측법검측miR-4634적Cq치위30.98±0.17,고우RNA추제법적29.22±0.21,차이유통계학의의(t=8.475,P＜0.01),이2충방법검측U6(직접검측법32.43±0.08,RNA추제법32.57±0.06;t=1.354,P＞0.05)、miR-24-1-5p(직접검측법20.73±0.14,RNA추제법21.06±0.21；t=1.749,P＞0.05)결과차이균무통계학의의.2충방법검측U6(r=0.860 5,P＜0.01)、miR-24-1-5p(r=0.728 9,P＜0.05)화miR-4634(r=0.748 5,P＜0.01)결과구유교호적상관성.혼합인물화단개인물역전록후PCR적Cq치지간무명현차이.결론 건립적혈장직접검측법지수대혈장표본진행간단적예처리,병가사용혼합인물진행역전록정량PCR,구유조작간편、검측성본저등우점,치득진일보추엄응용.
Objective To establish a new method for the detection of plasma miRNAs by real-time fluorescence quantitative PCR without RNA extraction.Methods 7.5 μ1 plasma separated from normal human blood was treated with equal amount of buffer containing Tween 20,which was used directly as sample for the following reverse transcription and PCR reaction to establish a direct assay for plasma nicroRNAs.U6 snRNA,miR-24-1-5p and miR-4634 were selected as target microRNAs for the determination of RT-qPCR.Both specificity and amplification efficiency were assessed.The difference of Cq values between traditional quantitative PCR detection method and our new method was also observed.Furthermore,the results of mixed primer or individual primer by RT-qPCR were compared in new established method.Results The amplification products of U6,miR-24-1-5p and miR-4634 show a single melting peak at 81 ℃,83 ℃ and 84.2 ℃,respectively,without printer dimer peak or non-specific peak in all 8 cases of healthy human plasma samples.Amplification efficiency for miR-24-1-5p reached 1.1 detected in plasma direct assay.The amplification curve of U6,miR-24-1-5p and miR-4634 is parallel in each sample,which demonstrated that the amplification of slope is consistent.The Cq value of miR-4634 determined by direct assay is higher than the result of the method by RNA extraction (plasma direct assay 30.98 ± 0.17,RNA extraction 29.22 ± 0.21 ;t =8.475,P ＜ 0.01),while there was no significant difference for the detection of U6 (plasma direct assay 32.43 ±0.08,RNA extraction 32.57 ±0.06;t =1.14,P ＞ 0.05) and miR-24-1-5p (plasma direct assay 20.73 ±0.14,RNA extraction 21.06 ±0.21 ;t =1.749,P ＞0.05) although they had significant correlation(r =0.860 5,0.728 9,0.748 5,P ＜ 0.05).No significant difference of Cq value between mixed primer reverse trauscription and individual primer reverse transcription.Conclusion A novel RT-qPCR direct assay of plasma miRNAs is established,which can improve the efficiency of plasma miRNAs determination in potential clinical applications.