Abi steponeplus system

Total RNA was extracted from mouse tissues using TRIzol reagent (Thermo Fisher Scientific Inc. Waltham, MA) and the first-strand cDNA was synthesized from 2.0 μg mRNA using High-Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific Inc. Waltham, MA). Real-time PCRprimers for mouse H6PDH (F: 5′-TGGCTACGGGTTGTTTTT GAA-3′; R: 5′-TATACACGGTACATCTCCTCTTCCT-3′), 11β-HSD1 (F: 5′-CCTTGGCC-TCATAGACACAGA AA C-3′; R: 5′-GGAGTCAAAGGCGATTTGTCAT-3′), ACC (F: 5′-TGT AAATCTGGCTGCATCCATTAT-3′; R: 5′-TGGTAGACTGCCCGTGTGA-3′), ATP-citrate lyase (ACL) (F: 5′ -ATGCCAAGACCATCCTCTCACT-3′; R: 5′-TCTCACAATGCCCTTGAAGGT-3′), HSL (F: 5′-GGGCAAAGAAGGATCGAAGAA-3′; R: 5′ GCGTAAATC CATGCTGTGTGA-3′), ATGL (F: 5′-TCGTGGATGTTGGTGGAGCT-3′; R: 5′-TGTGGCCTCATTCCTCCTA-3′) , C/EBPα (F: 5′-TGGACAAG AACAGCAACGAGTAC-3′; R: 5′-CGGTCATTGTCACTGGTCAACT-3′), and C/EBPβ (R:5′-CTGCGGGGTTGTTGATGT-3′; R: 5′-ATGCTCGAAACGGAAA GGT-3′) were designed by Primer express software 2.0 (Thermo Fisher Scientific Inc. Waltham, MA). Quantitative Real-time PCR analysis was performed with SYBER green kit following manufacturer’s protocol in the ABI StepOne Plus System (Thermo Fisher Scientific Inc. Waltham, MA). Threshold cycle (C_t) readings for each of the unknown samples were then used to calculate the amount of target genes. The expression for target genes was normalized to the 18S rRNA values.