国际检验医学杂志
國際檢驗醫學雜誌
국제검험의학잡지
INTERNATIONAL JOURNAL OF LABORATORY MEDICINE
2015年
1期
57-59
,共3页
实时荧光定量%血浆 microRNAs%外参照
實時熒光定量%血漿 microRNAs%外參照
실시형광정량%혈장 microRNAs%외삼조
real-time fluorescence quantitative%plasma microRNAs%exogenous reference
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。
目的：建立特異、穩定且可靠的血漿 microRNA（miRNAs）實時熒光定量 PCR 技術。方法收集10例健康人血漿標本，採用 mirVanaTM PARIS kit 試劑盒法提取血漿 miRNAs，以 miRNA 特異的莖環引物引導逆轉錄，通過 SYBR Green 實時熒光定量聚閤酶鏈反應（PCR）對外參照 cel-miR-39、cel-miR-238及血漿 miR-342-3p 進行檢測。結果健康人血漿 miR-342-3p 及外參 cel-miR-39、cel-miR-238可以實現特異性擴增及定量。溶解麯線顯示10例健康人血漿標本中 cel-miR-39、cel-miR-238和 miR-342-3p 擴增產物分彆在81．44、81．62、82．71℃時齣現單一峰，無引物二聚體峰和其他非特異峰齣現。血漿 miR-342-3p 批內批間重複性實驗的標準差在0．13～0．20、變異繫數（CV）在0．42％～0．66％，顯示該方法重複性較好。以 cel-miR-39、cel-miR-238作為外參照，同一血漿標本 miR-342-3p 的5次檢測結果ΔCt 的標準差為0．22、CV 為1．68％，說明 cel-miR0-39、cel-miR-238可作為血漿 miRNAs 實時熒光定量 PCR 檢測的穩定外參。結論實時熒光定量 PCR 技術可作為研究血漿 miRNAs 較好的技術平檯。
목적：건립특이、은정차가고적혈장 microRNA（miRNAs）실시형광정량 PCR 기술。방법수집10례건강인혈장표본，채용 mirVanaTM PARIS kit 시제합법제취혈장 miRNAs，이 miRNA 특이적경배인물인도역전록，통과 SYBR Green 실시형광정량취합매련반응（PCR）대외삼조 cel-miR-39、cel-miR-238급혈장 miR-342-3p 진행검측。결과건강인혈장 miR-342-3p 급외삼 cel-miR-39、cel-miR-238가이실현특이성확증급정량。용해곡선현시10례건강인혈장표본중 cel-miR-39、cel-miR-238화 miR-342-3p 확증산물분별재81．44、81．62、82．71℃시출현단일봉，무인물이취체봉화기타비특이봉출현。혈장 miR-342-3p 비내비간중복성실험적표준차재0．13～0．20、변이계수（CV）재0．42％～0．66％，현시해방법중복성교호。이 cel-miR-39、cel-miR-238작위외삼조，동일혈장표본 miR-342-3p 적5차검측결과ΔCt 적표준차위0．22、CV 위1．68％，설명 cel-miR0-39、cel-miR-238가작위혈장 miRNAs 실시형광정량 PCR 검측적은정외삼。결론실시형광정량 PCR 기술가작위연구혈장 miRNAs 교호적기술평태。
Objective To establish a specific,stable and reliable real-time fluorescence quantitative PCR for detecting plasma mi-croRNAs(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 spe-cific 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 amplifica-tion 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 sug-gesting 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 cel-miR-238.The SD and CV of ΔCt was 0.22,1.68%,re-spectively,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.
