Optimized RT-PCR method for assaying expression of monocyte chemotactic protein type 1 (MCP-1) in rabbit aorta.
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.
Sekalska
B
B
Department of Toxicology, Faculty of Food Sciences and Fisheries, Agricultural University of Szczecin, Papieza Pawła VI 3, 71-459, Szczecin, Poland. beata@tz.ar.szczecin.pl
Ciechanowicz
A
A
Dołegowska
B
B
Naruszewicz
M
M
D000818:Animals; D001011:Aorta / Q000737:chemistry*; D018932:Chemokine CCL2 / Q000235:genetics*; D015870:Gene Expression; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D008297:Male; D012333:RNA, Messenger / Q000032:analysis*; D011817:Rabbits; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018932:Chemokine CCL2; D012333:RNA, Messenger; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)