2.5 Complementary DNA synthesis and quantitative real-time–PCR

Complementary DNA (cDNA) was generated from 400 ng total RNA per sample using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher, UK) with random primers. cDNA samples were diluted 1:20 and real-time quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) and synthetic oligonucleotides as primers (see Supplementary material online, Table S1). Primers were designed to assay all annotated splice-variants of a gene where possible, and were checked for specificity using NCBI nucleotide BLAST. A six-point standard curve of two-fold dilution was prepared from pooled cDNA to assess amplification efficiency of primer pairs. All primers amplified with an estimated efficiency between 90% and 110%, and there was no evidence of inhibitors present in the reaction. Quantification was performed using the comparative Ct method.35 Target gene expression was normalized to the geometric mean of Gapdh and Sdha (cardiac tissue) and to 36b4 (subcutaneous and brown adipose tissue). The expression levels of the genes of reference did not differ between groups. Statistics were performed on the relative fold change (2^−ΔΔCT).