Analysis of leptin gene expression in chickens using reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.
Leptin is a peptide hormone product of the obese (ob) gene that functions in the regulation of appetite, energy expenditure and reproduction in animals and humans. We have developed a technique using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the analysis of chicken leptin (261 base pairs, bp) and beta-actin (612 bp) double-stranded DNA products from reverse transcription polymerase chain reaction (RT-PCR) assays. Amplicons were separated using a DB-1 coated capillary (27 cm x 100 microns I.D.) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethylcellulose (HPMC) in 1X TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microgram/ml EnhanCE fluorescent intercalating dye. RT-PCR samples (1-2 microliters) were diluted 1:100 with 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-CE-LIF to determine the effects of insulin and estrogen treatment on leptin gene expression relative to that of beta-actin in chicken liver and adipose tissue. In addition, we have constructed a chicken leptin mRNA competitor (234 bp amplicon) and evaluated it for use as an internal standard in the development of a quantitative-competitive RT-PCR assay. Our findings represent the first reported application of capillary electrophoresis to the analysis of leptin gene expression by RT-PCR.
Richards
M P
MP
US Department of Agriculture, Livestock and Poultry Sciences Institute, Beltsville, MD 20705-2350, USA. richards@lpsi.barc.usda.gov
Ashwell
C M
CM
McMurtry
J P
JP
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004247:DNA / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D004967:Estrogens / Q000494:pharmacology; D005453:Fluorescence; D015870:Gene Expression / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology; D007834:Lasers; D020738:Leptin / Q000032:analysis / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004967:Estrogens; D007328:Insulin; D020738:Leptin; D012313:RNA; D004247:DNA