RNA Extraction from Tissue Samples

Tissue samples, cut to 0.5 cm thickness on at least one side, were stored in RNAlater at 4°C for 2–7 days. RNA was recovered from tissue samples using a modification of the method described by Hansen et al., 2013 [65 (link)]. Briefly, up to 200 mg of tissue was disrupted in TRIzol (Lifetechnologies) with 2 x 5 mm stainless steel beads using the TissueLyser (Qiagen) for 3 minutes at 25 r/s twice. Following homogenization, samples in TRIzol were separated using Bromo-chloro-propane (Sigma). The aqueous phase was collected, and glycogen was added as a carrier. The samples were washed in isopropanol and ethanol precipitated. RNA was fully re-suspended in 5 mM tris pH 8.0.