RT-qPCR Validation of Gene Expression

Validations were carried out using RT-qPCR on inguinal WAT and iBAT from 12-week R6/2 mice and WT littermates. Further validations were then performed on 12- and 18-month Q175 mice and WT littermates. For RT-qPCR experiments, all samples were run in triplicate for each target gene and housekeeping gene, and relevant negative and positive controls were run on each plate. Melt curves were inspected for all assays, with the Tm checked to be within known specifications for each assay. Sample assay data points were included in data analysis only if detected with Ct < 37 and at least 3 Ct values lower than the corresponding negative control [27 (link)]. Any data that did not pass these criteria were omitted from all further analyses. Primers utilised for RT-qPCR validations (Table 1) were designed using QuantPrime [28 (link)] or PrimerQuest from Integrated DNA Technologies (PrimerQuest">http://eu.idtdna.com/PrimerQuest).