RNA isolation

The general procedures for RNA isolation and synthesis of cDNA were previously described by our group^51,52. For the current study, RNA was freshly isolated for all specimens from frozen tissue²⁹. Briefly, frozen tissue was dispersed using Brinkmann Polytron homogenizer in guanidinium thiocyanate (GTC) buffer (4–5 M GTC, 0.5% sodium-N-lauryl sarcosine, 10 mM EDTA, 20 mM Tris–HCl 0.1 M 2-mercaptoethanol). Homogenates were layered above 5.7 M CsCl/EDTA in diethyl pyrocarbonate (DEPC) H2O in polyallomer tubes (11 × 34 mm; Beckman, Indianapolis, IN). Using a TLS-55 swinging bucket rotor (Beckman, Indianapolis, IN) samples were centrifuged for 3 h at 55,000 rpm in a TL-100 ultracentrifuge (Beckman). Following centrifugation, excess GTC and CsCl were removed by aspiration and the RNA pellet was solubilized with RNA sample buffer (0.5% N-lauroylsarcosine, 5 mM EDTA, 5 mM tris, and 5% 2-mercaptoethanol). The RNA-buffer solution was transferred to tubes containing an equal volume of 50:50 phenol–chloroform (Calbiochem/EMD Millipore, San Diego, CA), vortexed, and centrifuged for 3 min at 13,000 rpm. The resulting supernatant was washed with 100% chloroform (Sigma-Aldrich, St. Louis, MO), centrifuged, and added to tubes containing 100% ETOH and 0.3 M sodium acetate. Following overnight incubation at -80 °C, RNA was precipitated by centrifugation for 20 min at 10,000 rpm and washed once with 80% ETOH. The resulting RNA pellet was re-suspended in DEPC H2O, and concentration was determined by ultraviolet absorbance spectroscopy (260 nm)^51,52.