[1]周 萍,杨玉琮,王 聪,等.miRNA-125a Taqman实时荧光定量PCR检测方法的建立及初步应用[J].现代检验医学杂志,2016,31(04):10-13.[doi:10.3969/j.issn.16717-414.2016.04.003]
　ZHOU Ping,YANG Yu-zong,WANG Cong,et al.Establishment and Preliminary Application of miRNA-125a Taqman Real-time Fluorescent Quantitative PCR Detection Method[J].Journal of Modern Laboratory Medicine,2016,31(04):10-13.[doi:10.3969/j.issn.16717-414.2016.04.003]
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miRNA-125a Taqman实时荧光定量PCR检测方法的建立及初步应用()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第31卷
期数:
2016年04期
页码:
10-13
栏目:
论著
出版日期:
2016-08-10

文章信息/Info

Title:
Establishment and Preliminary Application of miRNA-125a Taqman Real-time Fluorescent Quantitative PCR Detection Method
文章编号:
1671-7414(2016)04-010-04
作者:
周 萍; 杨玉琮; 王 聪; 胡 健; 陶 丹; 刘红莉
西安交通大学第一附属医院检验科,西安 710061
Author(s):
ZHOU Ping; YANG Yu-zong; WANG Cong; HU Jian; TAO Dan; LIU Hong-li
Department of Clinical Laboratory,the First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China
关键词:
miRNA;  肝癌;  Taqman 探针;  实时荧光定量PCR
分类号:
R735.7; Q503
DOI:
10.3969/j.issn.16717-414.2016.04.003
文献标志码:
A
摘要:
目的 建立一种用于肝癌早期诊断和监测的血清miRNA-125a Taqman实时荧光定量PCR检测方法。方法 采用Trizol法提取肝癌患者血清中总RNA,以肝癌发生具有代表性的miRNA-125a基因为检测序列,分别设计逆转录引物、扩增引物和Taqman探针; 构建和制备miRNA-125a重组质粒标准品,建立血清miRNA-125a Taqman 实时荧光定量PCR检测方法。结果 成功构建了miRNA-125a重组质粒载体,制备了miRNA-125a重组质粒标准品,建立了血清miRNA-125a Taqman实时荧光定量PCR扩增体系。经实验验证,所设计引物具有较好的特异性,引物的最低检测限为78.125 copies/μl; 对17例肝癌患者血清miRNA-125a的检测结果在(1.45×102～3.52×105)copies/μl之间。结论 血清miRNA-125a Taqman实时荧光定量PCR检测方法的建立,为原发性肝癌早期诊断和监测提供一种新的分子诊断定量方法。
Abstract:
Objective Tobuild the method of real-time fluorescence quota PCR examination of serum miRNA-125a Taqman for diagnosing and monitoring liver cancer early.Methods 1st,based on specific miRNA-125a geneassociated with liver cancer,to design reverse transcriptaseprimer,amplification primer and Taqman probeindividually through the Trizaol method to extract patients' total RNA from serum.2nd,to prepare the miRNA-125arecombinant plasmid standard substance and buildthe method of real-time fluorescence quota PCR examination of serum miRNA-125a Taqman.Results To form the miRNA-125a recombinant plasmid carriers successfully.To prepare the miRNA-125a recombinant plasmid standard substance successfully.To buildamplification system of real-time fluorescence quota PCR of serum miRNA-125a Taqman.It had been proved that the designed primer whose minimum detection limit was 78.125 copies/μl had proper specificity.The examination results of serum miRNA-125a of 17 patients with liver cancer vary between 1.45×102～3.52×105 copies/μl.Conclusion To setup the method of real-time fluorescence quota PCR examination of serum miRNA-125a Taqman can offer an efficientmolecular diagnosis way to diagnose and monitorthe primary liver cancer early.

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备注/Memo

备注/Memo:
基金项目:陕西省科技计划项目(2013K12-05-12),西安市卫生局科技计划项目(2013)资助。
作者简介:周 萍(1975-),女,本科,主管检验师,主要从事临床免疫和肿瘤诊断。
通讯作者:刘红莉(1965-),女,主任医师,主要从事分子免疫及感染疾病研究,E-mail:liuhonglili@sina.com。
更新日期/Last Update: 2016-08-10