应用实时荧光定量PCR检测血清miR-122和miR-22及其在慢性乙型肝炎患者中的表达

Detection of Serum miR- 122 and miR- 22 in Patients with Chronic Hepatitis B by Using of a Quantitative Real- time PCR

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 ×10^5、2 ×10^6、2 ×10^7 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.

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 ×10^5 , 2 ×10^6 , 2 ×10^7 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.