Relative mRNA levels were quantified using qPCR, adopted from previous reports.20 (link) Briefly, cDNA was synthesized from 400 ng total RNA via reverse transcription using Superscript III kit (Life Technologies) and qPCR assays were performed for each target and reference gene per sample with SsoAdvanced Universal SYBR Green Supermix (BioRad). All target and housekeeping gene primers based on sequences specific for Mus musculus were derived from PrimerBank.36 (link) Official gene symbols, primer sequences (5′ to 3′), respective housekeepers, product sizes, and PrimerBank IDs for target gene products are listed in Table 1; specifications of housekeeping genes used in this study (SDHA: succinate dehydrogenase complex, subunit A; Polr2b: polymerase [RNA] II [DNA directed] polypeptide B; Taf1b: TATA box binding protein [Tbp]-associated factor, RNA polymerase I) are summarized in Table 2. Housekeeper genes were selected based on stringent efficiency testing of each target-housekeeper pair. All primers had an annealing temperature of 60°C. Amplification efficiencies were tested for each primer pair, with efficiency (E) = [10(−1/S)] − 1, and relative quantification analysis of gene expression data was conducted according to the 2−∆∆CT method.37 (link)