Sekalska B, Ciechanowicz A, Dołegowska B, Naruszewicz M
Department of Toxicology, Faculty of Food Sciences and Fisheries, Agricultural University of Szczecin, Papieza Pawła VI 3, 71-459, Szczecin, Poland.
Biochem Genet. 2006 Apr;44(3-4):133-43. doi: 10.1007/s10528-006-9015-4. Epub 2006 Jun 20.
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 degrees 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.
逆转录聚合酶链反应（RT-PCR）是在基因结构和/或表达水平上开启了解动脉粥样硬化机制之路的最有用的分子生物学方法之一。我们针对温度曲线、产量与循环次数、基因组DNA对RNA模板的干扰以及重复性，对该技术进行了优化，以检测兔主动脉中单核细胞趋化蛋白1（MCP-1）基因的表达。还检测了组成型GAPDH基因表达的变异性。该研究在18只新西兰兔中进行，这些兔子被分为两组，分别喂食标准饲料2（S1）或3（S2）个月。实验结束时，取出兔升主动脉的一部分，从中分离RNA。特异性引物与MCP-1和GAPDH基因结合的最佳温度为63℃，MCP-1的PCR扩增最佳循环次数为22次，GAPDH为26次。无论有无逆转录酶，GAPDH扩增子大小均为465个碱基对，表明RNA模板被基因组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进行调整后的RT-PCR为检测兔主动脉中MCP-1基因的表达提供了一种可靠的方法。
