Total RNA was extracted from tissue samples (hypothalamus, liver, iBAT, and femur) using TRIzol reagent (Life Technologies) following the manufacturer’s instructions; 1 μg of total RNA was used to synthesize cDNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). An aliquot (1/100 vol) of the cDNA was then subjected to real-time quantitative PCR (qPCR) using iQ SYBR Green Supermix (Bio-Rad) in a 96-well real-time PCR machine (CFX96, Bio-Rad). Fold changes were calculated and determined using the 2-ΔΔCt method and expression levels normalized to the average of the housekeeping genes 18S or HRPT.

Primers used were as follows: mouse Pomc: F, 5′-CAGGTCCTGGAGTCCGAC-3′, R, 5′-CATGAAGCCACCGTAACG-3′; Npy: F, 5′-CTACTCCGCTCTGCGACACT-3′, R, 5′-AGTGTCTCAGGGCTGGATCTC-3′; Agrp: F, 5′-CGGCCACGAACCTCTGTAG-3′, R, 5′-CTCATCCCCTGCCTTTGC-3′; SF-1: F, 5′-CCCTTATCCGGCTGAGAATT-3′, R, 5′-CCAGGTCCTCGTCGTACGA-3′; ObRb: F, 5′-AACCCCAAGAATTGTTCCTGG-3′, R, 5′-GGAGACCATAGCTGCTGGGACC-3′; 5-HT2c: F, 5′-GCCAATGAACACCTTCTTTCC-3′, R, 5′-GCCAATTAGGTGCACAAGGAG-3′; Ucp1: F, 5′-GGCCCTTGTAAACAACAAAATAC-3′, R, 5′-GGCAACAAGAGCTGACAGTAAAT-3′; Pgc1α: F, 5′-AACCACACCCACAGGATCAGA-3′, R, 5′-TCTTCGCTTTATTGCTCCATGA-3′; Dio2: F, 5′-TTCTCCAACTGCCTCTTCCTG-3′, R, 5′-CCCATCAGCGGTCTTCTCC-3′; Tfam: F, 5′-AGGAGGCAAAGGATGATTCG-3′, R, 5′-CCTCAGGAGACAGATTTTTCCA-3′; Pklr: F, 5′-AGGAGTCTTCCCCTTGCTCTAC-3′, R, 5′-GGAGAGGCGTTTCAGGATATG-3′; PEPCK: F, 5′-CGCAAGCTGAAGAAATATGACAA-3′, R, 5′-TCGATCCTGGCCACATCTC-3′; G6Pase: F, 5′-TGGGCAAAATGGCAAGGA-3′, R, 5′-TCTGCCCCAGGAATCAAAAAT-3′; Ehhadh: F, 5′-CTTGGAATTCTGGATGTAG-3′, R, 5′-TGGGTTTACCTATAACCG-3′; PPARγ: F, 5′-CAAGAATACCAAAGTGCGATCAA-3′, R, 5′-GAGCTGGGTCTTTTCAGAATAATAAG-3′; Srebp1: F, 5′-GGAGCCATGGATTGCACATT-3′, R, 5′-GGCCCGGGAAGTCACTGT-3′; FASN: F, 5′-GGTGTGGTGGGTTTGGTGAATTGT-3′, R, 5′-TCACGAGGTCATGCTTTAGCACCT-3′; HSL: F, 5′-GCGCTGGAGGAGTGTTTTT-3′, R, 5′-CCGCTCTCCAGTTGAACC-3′; Rank: F, 5′-CCAGGAGAGGCATTATGAGCA-3′, R, 5′-ACTGTCGGAGGTAGGAGTGC-3′; Rankl: F, 5′-CAGCATCGCTCTGTTCCTGTA-3′, R, 5′-CTGCGTTTTCATGGAGTCTCA-3′; Trap: F, 5′-GCAACATCCCCTGGTATGTG-3′, R, 5′-GCAAACGGTAGTAAGGGCTG-3′; Dc-Stamp: F, 5′-GGGGACTTATGTGTTTCCACG-3′, R, 5′-ACAAAGCAACAGACTCCCAAAT-3′; Sp7: F, 5′-TCCCTGGATATGACTCATCCCT-3′, R, 5′-CCAAGGAGTAGGTGTGTTGCC-3′; Alp: F, 5′-CCTTGAAAAATGCCCTGAAA-3′, R, 5′-TTACTGTGGAGACGCCCATA-3′; 18S: F, 5′-AACCCGTTGAACCCCATT-3′, R, 5′-CCATCCAATCGGTAGTAGCG-3′; and HRPT: F, 5′-CTCATGGACTGATTATGGACAGGAC-3′, R, 5′-GCAGGTCAGCAAAGAACTTATAGCC-3′.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。