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United States Department of Agriculture

Agricultural Research Service

Title: Analysis of Leptin Gene Expression in Chickens Using Rt-Pcr and Capillary Electrophoresis with Laser-Induced Flurescence Detection

Authors
item Richards, Mark
item Ashwell, Christopher
item McMurtry, John

Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 27, 1999
Publication Date: N/A

Interpretive Summary: Leptin is a peptide hormone encoded by the obesity (ob) gene that functions in the regulation of feeding behavior, energy balance and reproduction in humans and animals. In mammals, the amount of leptin in blood increases in direct proportion to body fat mass. Circulating leptin signals the brain about the status of body energy (fat) stores, and the brain, in turn, activates specific neural pathways that modulate food intake and energy expenditure to help maintain energy stores at a set level. Thus, a negative feedback loop exists between adipose tissue and the central nervous system with leptin serving as an afferent signal to the brain. Recessive mutations in the genes encoding leptin and its receptor have been identified and linked with the pathological conditions of morbid obesity, diabetes and infertility. Therefore, it is important to be able to accurately monitor leptin expression at the levels of nucleic acid (mRNA) and protein. We are interested in studying the role of leptin in regulating food intake in domestic animals. Our objective in this study was to develop an analytical method, quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR),and apply it to the quantification of leptin gene expression in chickens. The QC-RT-PCR method developed in this study offers the most precise way currently available to monitor leptin expression in tissue samples obtained from chickens.

Technical Abstract: Leptin, a peptide hormone product of the obese (ob) gene, is known to function in the regulation of appetite and energy expenditure in animals and humans. We have developed a CGE-LIF technique for the analysis of chicken leptin (261 bp) and b-Actin (612 bp) dsDNA products from RT-PCR. Amplicons were separated using a DB-1 capillary (27 cm x 100 mm I.D.) at a field strength of 300V/cm in a replaceable sieving matrix consisting of 0.5% HPMC in 1X TBE with 0.5 mg/ml LiFluor dye (EnhanCE, Beckman Coulter). RT-PCR aliquots (1-2 ml) were diluted 1:100 in deionized water and introduced into the capillary by electrokinetic injection. Separations were completed in less than 6 min and the total time required per sample, including capillary conditioning, was 8 min. We have applied RT-PCR/CGE-LIF to determine the effects of insulin and estrogen treatments on leptin expression relative to that of b-Actin in chicken liver and adipose tissue. In addition, we have constructed a chicken leptin mRNA competitor (234 bp amplicon) and tested it for the development of a quantitative-competitive RT-PCR assay coupled with CGE- LIF. Our findings represent the first reported application of capillary electrophoresis to the analysis of leptin gene expression by RT-PCR.

Last Modified: 4/23/2014
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