RNA was extracted from adipose tissue with TRI Reagent combined with the RNeasy Mini Kit (Qiagen Ltd.), and from liver using RNeasy Mini Kit following manufacturer's instructions. On-column DNase digestion was performed using RNase-Free DNase set (Qiagen Ltd.), and RNA concentration and purity assessed using a NanoDrop One spectrometer (ThermoFisher Scientific, UK). Complimentary DNA was synthesised using TaqMan Reverse Transcription Kit (Applied Biosystems, UK) as described previously (Hogg et al., 2012) (link). To select the most stable housekeeping genes the geNorm Reference Gene Selection Kit (Primerdesign Ltd., UK) was used, identifying the suitability of the geometric mean of ACTB and MDH1 for liver and SAT, and RPS26 and 18S for VAT.
Primers (Supplementary Table 1) were designed and synthesised as described previously (Siemienowicz et al., 2020) (link). Quantitative RT-PCR was performed on 384-well plate format (Applied Biosystems) with all samples assayed in duplicate and housekeeping control genes included in each run, as well as template, RNA and RT-negative controls, using the ABI 7900HT Fast Real Time PCR system (Applied Biosystems) as described previously (Hogg et al., 2012) (link). The transcript abundance of target gene relative to the housekeeping genes was quantified using the Ct method (Livak and Schmittgen, 2001) (link).
Primers (Supplementary Table 1) were designed and synthesised as described previously (Siemienowicz et al., 2020) (link). Quantitative RT-PCR was performed on 384-well plate format (Applied Biosystems) with all samples assayed in duplicate and housekeeping control genes included in each run, as well as template, RNA and RT-negative controls, using the ABI 7900HT Fast Real Time PCR system (Applied Biosystems) as described previously (Hogg et al., 2012) (link). The transcript abundance of target gene relative to the housekeeping genes was quantified using the Ct method (Livak and Schmittgen, 2001) (link).