荧光定量RTPCR法检测OATPB和OATPD mRNA表达

山东大学学报(医学版) ›› 2009, Vol. 47 ›› Issue (7): 70-73.

荧光定量RTPCR法检测OATPB和OATPD mRNA表达

丁晓燕¹,王立新²，孔庆暖³，张日民³，王莉莉³，郭成浩¹

1. 山东大学医学院病理学教研室，济南 250012；
2. 江苏省淮安市第一人民医院，江苏淮安 223300；
3. 青岛市立医院病理科，山东青岛 266011

收稿日期:2008-03-17 发布日期:2009-07-16
通讯作者: 郭成浩(1965- )，男，教授，博士，主要从事实验病理学研究。 Email:chenghaoguo@sdu.edu.cn
作者简介:丁晓燕(1983- )，女，助教，硕士，主要从事实验病理学研究。

Fluorogenic quantitative RTPCR method in detection of organic anion transporting polypeptide(OATP B and OATPD)mRNA expressions

DING Xiaoyan¹, WANG Lixin ²,　KONG Qingnuan ³,　ZHANG Rimin ³, WANG Lili ³，　GUO Chenghao ¹

1. Department of Pathology, School of Medicine, Shandong University, Jinan 250012， China；
2. Department of Respiration, Huai′an First People′s Hospital, Huaian 223300, Jiangsu, China；
3. Department of Pathology, Qingdao Municipal Hospital, Qingdao 266011, Shandong, China

Received:2008-03-17 Published:2009-07-16

摘要/Abstract

摘要：

目的建立荧光定量RT—PCR检测肺癌及正常肺组织中人类有机阴离子转运多肽(organic anion transporting polypeptide, OATP) OATPB、OATPD mRNA表达的方法，了解肺癌和正常肺组织中OATPB、OATPD mRNA的表达水平。方法利用RTPCR方法从肺癌及正常肺组织中扩增出目的基因片段，用TA克隆的方法将基因片段插入到PGEMT质粒载体中，在LineGene荧光定量检测系统上建立检测OATPB、OATPD mRNA表达的标准曲线，并通过40例肺癌及正常肺组织标本进行验证。结果成功构建了OATP基因的质粒重组子，建立了在mRNA水平定量检测OATPB、OATPD基因的标准曲线，相关系数为0.998。以荧光定量RT—PCR法检测， OATPB、OATPD 的mRNA在肺癌及正常肺组织中均有表达，且肺癌中OATPB、OATPD mRNA的表达水平高于正常肺组织（P＜0．05）。结论成功建立了荧光定量RTPCR检测OATPB、OATPD mRNA表达的方法，检测结果用绝对拷贝数表示，定量准确可靠，敏感性高。

关键词: 逆转录聚合酶链反应；荧光定量；OATP基因；TA克隆

Abstract:

To establish a fluorogenic quantitative RTPCR (FQRTPCR) method for determining expressions of OATPB and OATPDmRNA, and to investigate expressions of OATPB and OATPDmRNA in lung neoplasms and nrmal lung tissues. MethodsThe OATP PCR product was subcloned into the PGEMT vector to construct recombinant plasmids. After optimizing the thermocycling conditions, a quantitative RTPCR assay was successfully developed to quantify OATP expression on the LineGene realtime PCR detection system, which was confirmed by 40 samples of lung cancer and normal lung tissues. ResultsThe standard curve was successfully established, and the linear regression value was0.998. Using FQRTPCR, we can get an accurate quantitative result of the OATPB and OATPDmRNA expressions. Moreover, expression levels of OATPB and OATPDmRNA in lung neoplasms were higher than those in normal lung tissues. ConclusionFQRTPCR for detecting expressions of OATPB and OATPDmRNA was sucessfully established, and is accurate and sensible.

Key words: Polymerase chain reaction； Fluorogenic quantitative； OATP gene; TA clone

中图分类号:

R361.3

. 荧光定量RTPCR法检测OATPB和OATPD mRNA表达[J]. 山东大学学报(医学版), 2009, 47(7): 70-73.

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