中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2011年
10期
926-930
,共5页
陈必成%王斯璐%白永恒%杨云秀%蔡勇%夏鹏%郑少玲%杨亦荣
陳必成%王斯璐%白永恆%楊雲秀%蔡勇%夏鵬%鄭少玲%楊亦榮
진필성%왕사로%백영항%양운수%채용%하붕%정소령%양역영
微小RNAs%T淋巴细胞%聚合酶链反应%筛选
微小RNAs%T淋巴細胞%聚閤酶鏈反應%篩選
미소RNAs%T림파세포%취합매련반응%사선
microRNA%T cells%Polymerase chain reaction%Scan
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.
目的 建立可同時篩選和定量檢測成熟型miR的熒光定量PCR方法.方法 改良通用莖環引物,在其莖環尾耑引入8箇隨機堿基片段,用于成熟型miR逆轉錄,以建立SYBR Green PCR篩選和定量檢測miR的方法(簡稱“改良通用莖環引物miR法”).同時,採用10倍梯度稀釋的miR-155標準品cDNA(1～109拷貝/μl)評價其靈敏度；採用鎔解麯線評價其檢測miR-155的特異性；通過對2×105、2×106、2×107拷貝/μl的miR-155標準品cDNA分彆進行批內20次重複實驗,以其循環閾值(Ct)的變異繫數(CV)評價其精密度；併與傳統莖環法進行比較,計算實驗所用時間和引物成本.然後,採用改良通用莖環引物miR法對植物血凝素(PHA)刺激培養0、16、24、48、72 h的人外週血T淋巴細胞內miR進行篩選(87箇靶miR,經PubMed檢索可能與免疫功能相關),併用以進行miR定量檢測分析.結果 建立的改良通用莖環引物法的最低檢測限為103拷貝/μl的miR-155 cDNA標準品；鎔解麯線于80℃呈單峰,證實為針對miR-155的特異性擴增；其批內重複實驗的精密度為CV＜2.5％.優化後的通用引物方法與傳統方法相比較,節約引物約75％(1 917 bp比7 851 bp),節省逆轉錄時間約120min(85 min比205 min).用改良通用莖環引物法篩選T淋巴細胞中87箇miR,85箇miR在PHA刺激前、後的Ct無變化,而miR-150和miR-155錶達量在T淋巴細胞激活前後分彆髮生超過10倍下降(72 h)和8倍的升高(48 h),且差異有統計學意義(Z=-2.023,P=0.043;Z=-2.032,P=0.042).結論 建立的改良通用莖環引物miR法具有快速、重複性好、靈敏、節約引物用量和實驗時間的優點,可用于T淋巴細胞的miR篩查和定量檢測.
목적 건립가동시사선화정량검측성숙형miR적형광정량PCR방법.방법 개량통용경배인물,재기경배미단인입8개수궤감기편단,용우성숙형miR역전록,이건립SYBR Green PCR사선화정량검측miR적방법(간칭“개량통용경배인물miR법”).동시,채용10배제도희석적miR-155표준품cDNA(1～109고패/μl)평개기령민도；채용용해곡선평개기검측miR-155적특이성；통과대2×105、2×106、2×107고패/μl적miR-155표준품cDNA분별진행비내20차중복실험,이기순배역치(Ct)적변이계수(CV)평개기정밀도；병여전통경배법진행비교,계산실험소용시간화인물성본.연후,채용개량통용경배인물miR법대식물혈응소(PHA)자격배양0、16、24、48、72 h적인외주혈T림파세포내miR진행사선(87개파miR,경PubMed검색가능여면역공능상관),병용이진행miR정량검측분석.결과 건립적개량통용경배인물법적최저검측한위103고패/μl적miR-155 cDNA표준품；용해곡선우80℃정단봉,증실위침대miR-155적특이성확증；기비내중복실험적정밀도위CV＜2.5％.우화후적통용인물방법여전통방법상비교,절약인물약75％(1 917 bp비7 851 bp),절성역전록시간약120min(85 min비205 min).용개량통용경배인물법사선T림파세포중87개miR,85개miR재PHA자격전、후적Ct무변화,이miR-150화miR-155표체량재T림파세포격활전후분별발생초과10배하강(72 h)화8배적승고(48 h),차차이유통계학의의(Z=-2.023,P=0.043;Z=-2.032,P=0.042).결론 건립적개량통용경배인물miR법구유쾌속、중복성호、령민、절약인물용량화실험시간적우점,가용우T림파세포적miR사사화정량검측.
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.
