Sensimix sybr green

Total RNA was isolated from randomly chosen subgroups of liver and BAT samples of the transplanted LDL receptor knockout recipients obtained at sacrifice, i.e. after the 20 week WTD challenge. cDNA was synthesized using RevertAid M-MuLV reverse transcriptase according to the manufacturer’s protocol (Thermo Scientific, USA). Relative mRNA expression levels were measured on a 7500 Fast Real-Time PCR system (Applied Biosystems) using SensiMix SYBR green technology. The average expression of the housekeeping genes GAPDH, HPRT and 36B4 was used as a reference for calculation of the relative expression levels of genes of interest. Primer sequences are available upon request.

RNA isolation and qRT-PCR analysis were performed as described previously^{8 (link),14 (link),50 (link)}. In short, RNA was isolated with a Machery-NucleoSpin RNA II kit (Bioké, Leiden, The Netherlands) according to the manufacturer’s instructions, and quantified using a Nanodrop ND-1000 (Wilmington, DE, USA). CDNA was prepared from total RNA using a cDNA Synthesis Kit (TAKARA BIO INC). Quantitative PCR was performed using Sensimix SYBRGreen (Applied Biosystems) or TaqMan (AXIN1; Hs00394718_m1, AXIN2; Hs00610344_m1, GAPDH; Hs02786624-g1) Gene Expression Assays (Applied Biosystems). Analyses were performed using the StepOne Real-Time PCR System and the StepOnev2.0 software (Applied Biosystems, Darmstadt, Germany). All expression levels are depicted relative to the expression of GAPDH. Primer sequences are provided in Supplemental Table S3.