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Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser‐induced fluorescence detection
Author(s) -
Richards Mark P.,
Ashwell Christopher M.,
McMurtry John P.
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000301)21:4<792::aid-elps792>3.0.co;2-m
Subject(s) - leptin , capillary electrophoresis , adipose tissue , real time polymerase chain reaction , microbiology and biotechnology , chemistry , gene expression , messenger rna , reverse transcription polymerase chain reaction , fluorescence , polymerase chain reaction , chromatography , biology , gene , endocrinology , biochemistry , obesity , physics , quantum mechanics
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 × 100 μm ID) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 × TBE (89 m M Tris‐base, 89 m M boric acid, 2 m M EDTA, pH 8.3) buffer with 0.5 μg/mL EnhanCE™ 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‐week‐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.