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order to determine the extent of Acadm mRNA expressed from the MCAD−/− mice, northern blot analysis was performed. Total RNA was isolated from heart, liver, brown fat, brain, kidney, skeletal muscle, white fat, and testes of 3-mo-old MCAD+/+ and MCAD−/− mice using the Ultraspec RNA Isolation Kit (BIOTEX Laboratories, Inc., Houston, Texas, United States) as per manufacturer's protocol. Ten μg of total RNA from each sample was loaded onto a 0.8% agarose-formaldehyde gel, transferred to nitrocellulose filter (Hybond N; GE Healthcare Amersham Biosciences Corp., Piscataway, New Jersey, United States), and hybridized with 32P-radiolabeled full-length mouse Acadm cDNA probe using standard procedures [28]. Hybridizations were performed under highly stringent conditions (42 °C in 2× SSC, 50% formamide, 10% dextran sulfate, 5× Denhardt's reagent, 1% SDS, and salmon sperm DNA) for 18 h. The hybridized filter was washed two times in 4× SSC, 0.1% SDS and two times in 2× SSC; 0.1% SDS at 55 °C for 1 h. The filter was exposed to autoradiographic film (Hyperfilm MP; GE Healthcare Amersham Biosciences, Piscataway, New Jersey, United States). Replicate agarose-formaldehyde gels were stained by ethidium bromide to verify equal RNA loading."}

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Total RNA was isolated from heart, liver, brown fat, brain, kidney, skeletal muscle, white fat, and testes of 3-mo-old MCAD+/+ and MCAD−/− mice using the Ultraspec RNA Isolation Kit (BIOTEX Laboratories, Inc., Houston, Texas, United States) as per manufacturer's protocol. Ten μg of total RNA from each sample was loaded onto a 0.8% agarose-formaldehyde gel, transferred to nitrocellulose filter (Hybond N; GE Healthcare Amersham Biosciences Corp., Piscataway, New Jersey, United States), and hybridized with 32P-radiolabeled full-length mouse Acadm cDNA probe using standard procedures [28]. Hybridizations were performed under highly stringent conditions (42 °C in 2× SSC, 50% formamide, 10% dextran sulfate, 5× Denhardt's reagent, 1% SDS, and salmon sperm DNA) for 18 h. The hybridized filter was washed two times in 4× SSC, 0.1% SDS and two times in 2× SSC; 0.1% SDS at 55 °C for 1 h. The filter was exposed to autoradiographic film (Hyperfilm MP; GE Healthcare Amersham Biosciences, Piscataway, New Jersey, United States). Replicate agarose-formaldehyde gels were stained by ethidium bromide to verify equal RNA loading."}