| 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 |
| 投稿时间：2015-01-29 修订日期：2015-02-10 点此下载全文 |
| 引用本文：姜跃炜,叶瑾,朱小区.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[J].医学研究杂志,2015,44(10):121-124 |
| DOI：
10.11969/j.issn.1673-548X.2015.10.034 |
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| 中文摘要: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 Ⅰ PCR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。 |
| 中文关键词:miR-122 miR-22 慢性乙型肝炎 |
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| Detection of Serum miR-122 and miR-22 in Patients with Chronic Hepatitis B by Using of a Quantitative Real-time PCR |
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| Abstract: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. Methods 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 coefficient 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, 2×107copies/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 patients. |
| keywords:miR-122 miR-22 Chronic hepatitis B |
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