Analysis of leptin gene expression in chickens using reverse transcriptionpolymerase chain reaction and capillary electrophoresis with laser-inducedfluorescence detection

Leptin is a peptide hormone product of the obese (ob) gene that functions i n the regulation of appetite, energy expenditure and reproduction in animal s and humans. We have developed a technique using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the analysis of chi cken leptin (261 base pairs, bp) and beta-actin (612 bp) double-stranded DN A products from reverse transcription polymerase chain reaction (RT-PCR) as says. Amplicons were separated using a DB-1 coated capillary (27 cmx100 mu m I.D.) at a field strength of 300 V/cm in a replaceable sieving matrix con sisting of 0.5% hydroxypropylmethylcellulose (HPMC) in 1X TEE (89 mM Tris-b ase, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 mu g/ml EnhanCE f luorescent intercalating dye. RT-PCR samples (1-2 mu l) were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic in jection. Separations were completed in less than 6 min and the total time r equired per sample, including capillary conditioning, was 8 min. We have ap plied RT-PCR-CE-LIF to determine the effects of insulin and estrogen treatm ent on leptin gene expression relative to that of beta-actin in chicken liv er and adipose tissue. In addition, we have constructed a chicken leptin mR NA competitor (234 bp amplicon) and evaluated it for use as an internal sta ndard in the development of a quantitative-competitive RT-PCR assay. Our fi ndings represent the first reported application of capillary electrophoresi s to the analysis of leptin gene expression by RT-PCR. Published by Elsevie r Science B.V.