CAJ | 학술논문

目的建立血清中microRNA( miRNA)的实时荧光定量PCR( qRT-PCR)的检测方法,对慢性乙型肝炎患者血清中miR-122和miR-22的表达水平进行检测和分析,探讨其在慢性乙型肝炎患者血清中表达的意义. 方法茎环引物用于成熟型miRNA反转录,以建立SYBR Green ⅠPCR筛选和定量检测miRNA的方法. 同时,采用10倍梯度稀释的miR-122标准品cDNA(1~109 拷贝/微升)评价其敏感度;采用熔解曲线评价其检测miRNA的特异性;通过对2 ×105、2 ×106、2 ×107 拷贝/微升的miR-122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. 采用建立的qRT-PCR技术检测慢性乙型肝炎患者及正常人血清中miR-122和miR-22的表达水平. 结果建立了茎环引物的RT-PCR法检测血清中的miRNA,该方法的线性范围宽,敏感度高,重复性好,方法简便. 在慢性乙型肝炎患者组中血清miR-122和miR-22的相对表达量分别为17.88和5.35;与正常对照组中血清miR-122和miR-22的相对表达量(分别为1.80和1.67)比较,差异有统计学意义( P均=0.000). 结论建立了茎环引物RT-PCR实时荧光定量PCR方法检测血清中的miR-122和miR-22,血清miR-122和miR-22在慢性乙型肝炎患者的血清中表达显著升高.
목적 건립혈청중microRNA( miRNA)적실시형광정량PCR( qRT-PCR)적검측방법,대만성을형간염환자혈청중miR-122화miR-22적표체수평진행검측화분석,탐토기재만성을형간염환자혈청중표체적의의. 방법 경배인물용우성숙형miRNA반전록,이건립SYBR Green ⅠPCR사선화정량검측miRNA적방법. 동시,채용10배제도희석적miR-122표준품cDNA(1~109 고패/미승)평개기민감도;채용용해곡선평개기검측miRNA적특이성;통과대2 ×105、2 ×106、2 ×107 고패/미승적miR-122표준품cDNA분별진행비내20차중복실험,이기순배역치( Ct)적변이계수( CV)평개기정밀도. 채용건립적qRT-PCR기술검측만성을형간염환자급정상인혈청중miR-122화miR-22적표체수평. 결과 건립료경배인물적RT-PCR법검측혈청중적miRNA,해방법적선성범위관,민감도고,중복성호,방법간편. 재만성을형간염환자조중혈청miR-122화miR-22적상대표체량분별위17.88화5.35;여정상대조조중혈청miR-122화miR-22적상대표체량(분별위1.80화1.67)비교,차이유통계학의의( P균=0.000). 결론 건립료경배인물RT-PCR실시형광정량PCR방법검측혈청중적miR-122화miR-22,혈청miR-122화miR-22재만성을형간염환자적혈청중표체현저승고.
Objective To establish a real -time quantitative PCR ( RT-PCR) assay for detecting serum miR -122, miR-22, and evaluate the clinical significance of miR -122 and miR-22 in patients with chronic hepatitis B ( CHB) by using of this assay .Meth-ods The mature miRNAs were reversely transcripted by using of stem -loop primers .SYBR GreenⅠquantitative real-time PCR ( qRT-PCR) was used for quantification of the miRNAs .The sensitivity of this assay was evaluated by using of the 10-fold-diluted miRNA-122 cDNA standards and the specificity was verified by using of melting curve assay .The accuracy was assessed by intra -assay coeffi-cient of variation (CV) of threshold cycle (Ct value), which were calculated from a 20-times-repeat detection of the miR -122 cDNA (2 ×105 , 2 ×106 , 2 ×107 copies/μl) standards.Using the established qRT -PCR assay, we detected the expression of serum miR -122 and miR-22 in the patients with CHB and healthy controls .Results The qRT-PCR assay exhibited good performances in the linear range, sensitivity and reproducibility while detecting miR -122 and miR-22.The relative level of miR -122 and miR-22 was 17.88 vs 5.35 in the CHB patients and 1.80 vs 1.67 in the controls (P=0.000).Conclusion Using of stem-loop primers, we established a qRT-PCR assay for detection of serum miR -122 and miR-22.Serum miR-122 and miR-22 increased significantly in the CHB pa-tients.