Quantitative analysis of leptin mRNA using competitive reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection

Leptin, the protein hormone product of the obese (ob) gene, functions in th e regulation of appetite, energy expenditure, and reproduction in animals a nd humans. Since changes in the level of circulating leptin can have marked physiological consequences, it is important to be able to accurately quant ify 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 pol ymerase chain reaction (QC-RT-PCR) assay for leptin mRNA. Capillary electro phoresis with laser-induced fluorescence detection (CE-LIF) was utilized fo r the separation and analysis of chicken leptin target (261 bp) and competi tor (234 bp) dsDNA products from QC-RT-PCR assay samples. Leptin amplicons were separated using a DB-1 coated capillary (27 cm X 100 mu m ID) at a fie ld strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 x TEE (89 mM Tris-base, 89 mM bor ic acid, 2 mM EDTA, pH 8.3) buffer with 0.5 mu g/mL EnhanCE(TM) fluorescent intercalating dye. Samples were diluted 1:100 with deionized water and int roduced into the capillary by electrokinetic: injection. QC-RT-PCR/CE-LIF w as 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 quanti tative analysis of leptin gene expression using QC-RT-PCR/CE-LIF.