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 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 microm ID) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethyl cellulose (HPMC) in 1 x TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microg/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.
Richards
M P
MP
U.S. 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
D000273:Adipose Tissue / Q000737:chemistry; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D007202:Indicators and Reagents; D007834:Lasers; D020738:Leptin / Q000235:genetics*; D008099:Liver / Q000737:chemistry; D008297:Male; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence
D007202:Indicators and Reagents; D020738:Leptin; D012333:RNA, Messenger