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首页> 外文期刊>Biochemical Genetics >Optimized RT-PCR Method for Assaying Expression of Monocyte Chemotactic Protein Type 1 (MCP-1) in Rabbit Aorta
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Optimized RT-PCR Method for Assaying Expression of Monocyte Chemotactic Protein Type 1 (MCP-1) in Rabbit Aorta

机译：优化RT-PCR方法测定兔主动脉中单核细胞趋化蛋白1型（MCP-1）的表达

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摘要

The reverse transcription polymerase chain reaction (RT-PCR) is one of the most useful molecular biology methods in opening the way to understanding of the mechanisms of atherosclerosis on the gene structure and/or expression level. We optimized this technique for assaying expression of the monocyte chemotactic protein type 1 (MCP-1) gene in rabbit aorta with respect to the temperature profile, yield to cycle number, interference of genomic DNA with the RNA matrix, and repeatability. Variability of expression of the constitutive GAPDH gene was also examined. The study was done in 18 New Zealand rabbits allocated to two groups and fed a standard chow for 2 (S1) or 3 (S2) months. The experiment ended with removal of part of the ascending rabbit aorta, from which RNA was isolated. The optimal temperature for binding of specific primers to the MCP-1 and GAPDH genes was 63°C, and the optimal number of cycles for PCR amplification was 22 for MCP-1 and 26 for GAPDH. The GAPDH amplicon size was 465 base pairs in the presence or absence of reverse transcriptase showing contamination of the RNA matrix with genomic DNA. Repeatability of the RT-PCR method was 8.7%, and variability of expression of the GAPDH gene was 7.7%. Thus, RT-PCR adjusted for contaminating genomic DNA provides a reliable way of assaying expression of the MCP-1 gene in rabbit aorta.
机译：逆转录聚合酶链反应（RT-PCR）是在了解基因结构和/或表达水平上的动脉粥样硬化机理方面最有用的分子生物学方法之一。我们优化了此技术，用于分析兔的主动脉中单链趋化蛋白1型（MCP-1）基因在温度分布，循环产量，基因组DNA对RNA基质的干扰以及可重复性方面的表达。还检查了组成型GAPDH基因的表达变异性。这项研究是在18只新西兰兔子中进行的，每只兔子分为两组，每只标准松鼠喂养2（S1）或3（S2）个月。实验结束时，移除了一部分升主动脉，从该主动脉中分离出RNA。特异性引物与MCP-1和GAPDH基因结合的最佳温度为63°C，对于MCP-1而言，PCR扩增的最佳循环数为22，对于GAPDH为26。在存在或不存在逆转录酶的情况下，GAPDH扩增子大小为465个碱基对，显示RNA基质被基因组DNA污染。 RT-PCR方法的重复性为8.7％，GAPDH基因表达的变异性为7.7％。因此，针对污染的基因组DNA进行了调整的RT-PCR提供了一种可靠的方法来测定兔主动脉中MCP-1基因的表达。

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  • 来源
    《Biochemical Genetics》 |2006年第4期|129-139|共11页
  • 作者

    B. Sękalska; A. Ciechanowicz; B. Dołęgowska; M. Naruszewicz;

  • 作者单位

    Department of Toxicology Faculty of Food Sciences and Fisheries Agricultural University of Szczecin Papieża Pawła VI 3 71-459 Szczecin Poland;

    Clinical Biochemistry and Laboratory Diagnostics Independent Laboratory of Patobiochemistry and Molecular Biology Pomeranian Medical University Al. Powstańców Wlkp. 72 70-111 Szczecin Poland;

    Department of Biochemistry and Chemistry Pomeranian Medical University Al. Powstańców Wlkp. 72 70-111 Szczecin Poland;

    Clinical Biochemistry and Laboratory Diagnostics Pomeranian Medical University Al. Powstańców Wlkp. 72 70-111 Szczecin Poland;

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  • 正文语种 eng
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  • 关键词

    polymerase chain reaction; reverse transcription; monocyte chemotactic protein type 1; atherosclerosis;

    机译：聚合酶链反应;逆转录;单核细胞趋化蛋白1型;动脉粥样硬化;

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