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United States Department of Agriculture

Agricultural Research Service

Title: Quantitative Analysis of Leptin Mrna Using Competitive Reverse Transcription Polymerase Chain Reaction & Capillary Electrophoresis with Laser-Induced Fluorescence Detection

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

Submitted to: Electrophoresis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 2, 1999
Publication Date: N/A

Interpretive Summary: Leptin is a peptide hormone encoded by the obese (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 a 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 gene expression at the level of nucleic acid (mRNA). 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, the protein hormone product of the obese (ob) gene, functions in the regulation of appetite, energy expenditure and reproduction in animals and humans. Since changes in the level of circulating leptin can have marked physiological consequences, it is important to be able to accurately quantify leptin gene expression. Toward this goal, we have constructed a chicken leptin RNA competitor and successfully employed it as an internal standard in the development of a quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR) assay for leptin mRNA. Capillary electrophoresis with laser-induced fluorescence detection (CE- LIF) was utilized for the separation and analysis of chicken leptin target (261 bp) and competitor (234 bp) dsDNA products from QC-RT-PCR assay samples. Leptin amplicons were separated using a DB-1 coated capillary (27 cm x 100 mm I.D.) at a field strength of 300V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethylcellulose (HPMC) in 1X TBE (8 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 mg/ml EnhanCETM fluorescent intercalating dye. Samples were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. QC-RT-PCR/CE-LIF was used to quantify leptin mRNA in liver and adipose tissue from 8 wk old male and female broiler chickens. This study is the first report of quantitative analysis of leptin gene expression using QC-RT-PCR/CE-LIF.

Last Modified: 4/19/2014
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