Applied biosystems 7900

Total RNA from livers and hepatocytes was extracted and reverse‐transcribed as described previously (Chang et al. 2010, 2012). cDNA was analyzed by quantitative real‐time PCR analysis using a SYBR green PCR kit (Bio‐Rad, Hercules, CA) and an Applied Biosystems 7900 (Applied Biosystems, Carlsbad, CA). Relative mRNA expression of the genes of interest was determined after normalization to cyclophilin by the ΔΔCt method.

Brain or liver samples collected were lysed and homogenized in 350 μl of lysis buffer (RLT Buffer, Qiagen) using the Fast prep 24 lyzer (MPbio, Luzern, Switzerland) according to the manufacturer’s instructions. Total RNA was isolated on spin columns with silica-based membranes (RNeasy Mini Kit, Qiagen, Basel, Switzerland), following the manufacturer’s instructions. RNA was eluted with 30 μl of H₂O. 200 ng of purified RNA was reverse transcribed in a volume of 50 μl using the RT High Capacity RNA-to-cDNA Kit (Applied Biosystems, Rotkreuz, Switzerland). Quantitative real-time PCR analysis was performed on cDNA obtained with the Applied Biosystems 7900 (Applied Biosystems, Rotkreuz, Switzerland) Real-Time PCR System using Power SYBR Green Taq polymerase master mix (Applied Biosystems, Rotkreuz, Switzerland). Primer sequences used for mRNA quantification were directed against NPY, AgRP, POMC, CART, BHBDH, HMGcs2, PEPCK, G6Pase, and Glycogen Phosphorylase mRNAs as well as β−2-microglobulin mRNA used as an endogenous control (See Table 1 for sequences). Data were analyzed with RQManager 1.2 software (Applied Biosystems, Rotkreuz, Switzerland) for relative quantitation of gene expression (RQ) using the 2^−ΔΔCT method58 (link).

RNA was extracted from tumor cells with RNeasy Plus Mini Kit (Qiagen), followed by quantification with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions. cDNAs were synthesized with random hexonucleotide primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen). RT-PCR was performed with Power SYBR Green Master Mix (Invitrogen) as the fluorescent dye and gene specific primers using Applied Biosystems 7900 (Applied Biosystems, Grand Island, NY). No template controls were employed for detecting nonspecific amplification. The sequences of RT-PCR primers were: 5′-CGACCACAAGGCCCTCAGTA-3′ (forward) and 5′-CAGCCTTGGTGTTGGAGGAG-3′ (reverse) for N-Myc; 5′-GGATTTTTTTCGGGTAGTGGAA-3′ (forward) and 5′-TTCCTGTTGGTGAAGCTAACGTT-3′ (reverse) for c-Myc; 5′- GATCCAGACTCGCACTGGAC-3′ (forward) and 5′- CCCGAGGTCAGAGGTTTGTT-3′ (reverse) for JMJD6; 5′-GGCCAAGAACAACATCCAGT-3′ (forward) and 5′-CGTGTTCATCAGCTCCTTCA-3′(reverse) for E2F2; and 5′-AGGCCAACCGCGAGAAG-3′ (forward) and 5′-ACAGCCTGGATAGCAACGTACA-3′ (reverse) for Actin. The primers were synthesized by Sigma (Sigma, Sydney, Australia). The comparative threshold cycle (△△Ct) method was employed to quantify fold changes in the expression of target genes^{34 (link)}, relative to the housekeeping gene actin.

To validate differential expression of genes that are enriched in the biological pathways identified by pathway over-representation analysis of 230 differentially expressed fetal BAT genes (nominal p-value ≤0.05 and absolute fold-change ≥1.5) and 302 differentially expressed placental genes (nominal p-value ≤0.05), we selected top 2 pathways per tissue based on their high significance levels. 1 μg of RNA samples was reverse transcribed using oligo dT primers and M-MLV reverse transcriptase (Promega), and 4 ng of cDNA were amplified by specific primers in 10ul reaction using SYBR Green Supermix (Bio-Rad) on an Applied Biosystems 7900 (Applied Biosystems). For each gene, relative abundance of mRNA was determined after normalization to cyclophilin by the 2^-ΔΔCt method.

Total RNA from tissues or cells was reverse-transcribed for quantitative real-time PCR analysis as described previously (Chang et al., 2010 (link), 2012 (link)). Gene expression analysis was carried out using the Applied Biosystems 7900 (Applied Biosystems) and iTaq Universal SYBR Green Supermix (Bio-Rad). Relative mRNA expression of the genes of interest was determined using gene-specific primers after normalization to cyclophilin by the 2^-ΔΔCt method. Primer sequences were obtained from the PrimerBank public resource (Wang and Seed, 2003 (link)). Sgk2 fwd: 5'- CCAATGGGAACATCAACC-3'; Sgk2 rev: 5'-CAGTAGGACCTTCCCGTAGT-3'.