Sigma genelute mammalian total rna kit

Total RNA was extracted from a total of 37 tissues using TRIzol. Specifically, we extracted RNA from fifteen livers from males and from 22 livers from females. For RNA extractions, we removed each sample from -80°C long-term storage and incubated them on ice to thaw. Using a pipette, the RNALater solution was removed. We added 1ml of autoclaved phosphate-buffered saline to each tissue to briefly wash them before using sterilized forceps to transfer each washed tissue to an individual 2ml screw-cap homogenization tube. To each sample, 200ul of TRIzol was added and the sample was transferred to -80°C storage. Tissue disruption was performed using a 2 mm steel bead and a Tissuslyer II (Qiagen, UK). To each sample, a 2 mm steel bead was added and samples were homogenized at 30 Hz for 30 s. Post-homogenization, samples were visually inspected to ensure thorough disruption. Total RNA was extracted using chloroform followed by isopropanol precipitation. Precipitated RNA was washed using three washes of ethanol before elution in the elution buffer (Sigma, UK). Total RNA was purified using the Sigma GenElute Mammalian Total RNA kit. Quality assessment was initially performed using a NanoDrop ND-1000 (ThermoFisher, UK) while an accurate assessment of quantity was estimated using the Qubit fluorometer, followed by a TapeStation 2200 (Agilent, UK).