RNA isolation from cells

Prep Cells

 * Aspirate media
 * Wash cells in ice-cold PBS
 * Aspirate PBS

Isolate RNA

 * Add 350/600uL RLT (prepare fresh lysis buffer and add 100X B-ME)
 * Scrape cells off dish and transfer to RNase-free tube
 * Sit on ice 3-5’
 * Keep samples on ice until all are processed
 * Homogenize samples by vortex 1’
 * Pipette sample into purple-top QIAshredder column
 * Centrifuge 2’ at max speed and RT
 * OPTIONAL: Pass lysate through a gDNA Eliminator column (this replaces DNase treatment after RNA isolation—if columns are available, do this step)
 * Add 350/600uL 70% EtOH and mix
 * Transfer 700uL to pink-top RNAeasy spin column
 * Centrifuge 1’ at max speed and RT
 * Discard flow through
 * If using 600ul lysate:
 * Transfer remaining ~600uL to pink-top RNeasy spin column
 * Centrifuge 1’ at max speed and RT
 * Discard flow through
 * Add 700uL RW1 buffer
 * Centrifuge 1’ at max speed and RT
 * Pour off flow-through
 * Add 500uL RPE buffer
 * Centrifuge 1’ at max speed and RT
 * Pour off flow-through
 * Repeat RPE wash
 * Centrifuge 1’ at max speed at RT with no wash to dry out column
 * Place column in a new RNase-free tube
 * Add 30-50uL RNase-free water to center of column
 * Sit at RT for 1-5’
 * At RT, centrifuge samples 1’ at max speed to elute RNA
 * Measure RNA concentration using Nanodrop
 * Keep samples on ice; store samples at -80°