Semi-quantification of human C-C chemokine mRNAs with reverse transcription/real-time PCR using multi-specific standards

被引：38

作者：

Dumoulin, FL ^{[1
]}

Nischalke, HD ^{[1
]}

Leifeld, L ^{[1
]}

von dem Bussche, A ^{[1
]}

Rockstroh, JK ^{[1
]}

Sauerbruch, T ^{[1
]}

Spengler, U ^{[1
]}

机构：

[1] Univ Bonn, Dept Med 1, D-53127 Bonn, Germany

来源：

JOURNAL OF IMMUNOLOGICAL METHODS | 2000年 / 241卷 / 1-2期

关键词：

C-C chemokines; mRNA quantification; real-time polymerase chain reaction; liver disease; viral hepatitis;

D O I：

10.1016/S0022-1759(00)00210-6

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A reverse transcription/real-time polymerase chain reaction (PCR) assay was established to semi-quantify the mRNA levels of the human C-C chemokines RANTES, MIP-1 beta and MCP-1 relative to the housekeeping gene beta-actin. The assay showed a high sensitivity (below 60 cDNA molecules/10 mu l reaction) and dynamic range (8 log units); both within-assay and inter-assay variability were below 0.06 log units and the accuracy was +/-0.06 log units for all four chemokines. Moreover, it is demonstrated that a multi-specific DNA fragment, which had previously been constructed for competitive PCR, can be used as a reliable external standard. This allows a direct semi-quantitative comparison of different chemokine mRNA levels and is a convenient alternative to the use of different sets of homologous external standards. The method was successfully applied to the semi-quantification of chemokines in human liver specimens and should be useful in further studies on steady state mRNA levels of C-C chemokines from low cell numbers or small tissue specimens. (C) 2000 Elsevier Science BN. All rights reserved.

引用

页码：109 / 119

页数：11

共 32 条

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