Fresh human skeletal muscle was obtained from tissue surrounding a surgically removed tumor of the quadriceps femoris. Total cellular RNA was isolated from cultured human fibroblasts, myoblasts, ARPE19 cells, and fresh muscle using an RNA isolation kit (RNeasy Mini; Qiagen Sciences, Valencia, CA). The concentration of the extracted RNA was determined spectrophotometrically, and 500 ng total RNA was used for each RT reaction. cDNA templates for PCR were prepared with random primers and M-MLV RT (Promega, Madison, WI). The resultant cDNA (diluted 1:5) was used for PCR reaction carried out for 2 minutes at 50°C and for 10 minutes at 95°C, followed by 44 cycles for 15 seconds at 95°C and for 1 minute at 60°C. Quantitative real-time PCR (QRT-PCR) was performed on a detection system (ABI PRISM 7000 Sequence Detection System; Applied Biosystems, Foster City, CA) and predesigned, gene-specific probe and primer sets for human CPT1A, CPT1B, and CPT1C (TaqMan; Assays-on-Demand; Applied Biosystems). Primer sets included the following primers: 5′-AAACGGCCAACTGCATGTCCAGCCA-3′ (Hs00157079_m1) for CPT1A, covering both transcriptional variants, 5′-TGTCATGGACCTTGTGCTCATCAAG-3′ (Hs00189258_m1) for CPT1B, covering all four transcriptional variants, and 5′-GTCCAATTATGTCAGTGACTGGTGG-3′ (Hs00380581_m1) for CPT1C (Hs00380581_m1).
Expression of mRNA was assessed by evaluating threshold cycle (C
T) values in quadruplicate reactions. Values were normalized against the expression level of a house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (
GAPDH). Gene expression levels of each
CPT1 isoform were evaluated by comparing their expression with that in fibroblasts using the comparative 2
−ΔΔC T method of quantification.
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