Mammary Tissue RNA Extraction Protocol

For total RNA extraction, snap-frozen mammary tissues were transferred to 2-ml tubes containing ceramic beads (OMNI International) in the presence of 0.5 ml Trizol (Bio-Rad) and were homogenized using an Omni Bead Ruptor 24 (OMNI International). The samples were lysed by running 2 cycles at the speed of 5.65 m/sec for 45 s, with a 30-s interval between each cycle. The tissue homogenates were spun at 10,000 rpm for 5 min at 4 °C, and the supernatant was processed using a Trizol (Bio-Rad) extraction method to extract RNA. To study mRNA levels, first-strand cDNA was synthesized using an iScript kit (Bio-Rad) as described previously [15 (link)]. mRNA expression of HMGCR, AMPK, and reference control -ribosomal protein L19 was measured by SYBR green-based chemistry (Bio-Rad) using the delta Ct (ΔΔCt) method as described before [15 (link), 18 (link), 19 (link)]. The expression levels of the genes of interest were measured by normalizing their signal (Ct) relative to loading control L19 and calculating the fold change in treated samples compared to vehicle control as the reference. The primer sequences for detecting mouse transcripts are provided in Supplementary Table 1.