RNA extraction and quality control

Total RNA was extracted using the PureLink RNA Mini Kit (Life Technologies, Carlsbad), following the manufacturer’s recommendations. Briefly, excess RNA later was removed by pipetting, and sample homogenization on lysis buffer was also carried out by pipetting samples up and down for about 60 times. Each sample was eluted into 35μL of RNAse free water.

Sample concentration was measured by Nanodrop spectrophotometer (Nano Drop Technologies Inc, Wilmingtom; ND-1000), and RNA quality was assessed by Bioanalyzer (Agilent Technologies Inc, Santa Clara, CA; 2100 Bioanalyzer), using the Agilent RNA 6000 Nano kit (Agilent Technologies) and following the manufacturer’s recommendations. Only one out of the forty eight samples displayed RIN (RNA integrity number) value lower than 7 (Replicate 3–14d Pi–RIN 6.7).