一种用于检测组织因子信使核糖核酸的定量实时聚合酶链反应方法。

A quantitative real-time PCR method for tissue factor mRNA.

作者信息

Mälarstig Anders, Tenno Taavo, Jossan Surinder, Aberg Mikael, Siegbahn Agneta

机构信息

Department of Medical Sciences, Uppsala University, Uppsala S-75185, Sweden.

出版信息

Thromb Res. 2003;112(3):175-83. doi: 10.1016/j.thromres.2003.11.001.

DOI:10.1016/j.thromres.2003.11.001

PMID:14967415

Abstract

BACKGROUND

Tissue factor (TF) is primarily known for its function to initiate blood coagulation. The range of in vivo expression of TF is wide and requires a dynamic assay for monitoring. A general method for TF mRNA quantitation that is dynamic, sensitive and applicable to a variety of experimental systems or clinical situations is therefore desirable.

OBJECTIVES

To develop a method for sensitive and dynamic quantitation of TF mRNA in human blood cells.

METHODS

TF mRNA expression was analysed and evaluated in monocyte isolations, in whole blood (healthy volunteers and patients scheduled for percutaneous coronary intervention, PCI) and in a panel of human cell lines. RNA was extracted, reverse transcribed and subjected to real-time PCR amplification, according to the TaqMan technology. A TF plasmid was constructed as calibrator of the assay. Two housekeeping genes used as endogenous controls for cDNA quality and integrity were evaluated.

RESULTS

The assay was linear by seven orders of magnitude and detected down to 10(2) copies of the TF plasmid. The coefficient of variation was 4% intra-assay and 28% between the assays when using beta2MG as endogenous control. The beta-actin gene expression was induced by treatment with lipopolysaccharide (LPS) in blood leukocytes and could not be used as an endogenous control. However, beta2MG showed only minor variations upon treatment with LPS. The TF mRNA and antigen expression, measured in a Western blot, correlated well (R(2)=0.903) in a panel of 11 human cell lines.

CONCLUSIONS

We have established a method for sensitive and dynamic quantitation of TF mRNA in experimental systems and for clinical situations.

摘要

背景

组织因子（TF）主要因其启动血液凝固的功能而闻名。TF在体内的表达范围广泛，需要动态检测来进行监测。因此，需要一种动态、灵敏且适用于各种实验系统或临床情况的TF mRNA定量通用方法。

目的

开发一种灵敏且动态定量人血细胞中TF mRNA的方法。

方法

在单核细胞分离物、全血（健康志愿者和计划进行经皮冠状动脉介入治疗的患者）以及一组人细胞系中分析和评估TF mRNA的表达。根据TaqMan技术提取RNA、进行逆转录并进行实时PCR扩增。构建TF质粒作为检测的校准物。评估了两个用作cDNA质量和完整性内参的管家基因。

结果

该检测在七个数量级上呈线性，可检测低至10²拷贝的TF质粒。以内源性对照β2微球蛋白（β2MG）进行检测时，批内变异系数为4%，批间变异系数为28%。β-肌动蛋白基因的表达在血液白细胞中经脂多糖（LPS）处理后被诱导，不能用作内源性对照。然而，β2MG在LPS处理后仅显示出微小变化。在11种人细胞系中，通过蛋白质印迹法测量的TF mRNA和抗原表达相关性良好（R² = 0.903）。