4.3. RNA Extraction

Total RNA isolation was carried out using the modified Chomczynski method with the TRI Reagent (Sigma, Saint Louis, MO, USA) and RNeasy Mini Elute cleanup Kit (Qiagen, Hilden, Germany), according to the manufacturer’s guidelines. The concentration of the total RNA was determined spectrophotometrically by measuring absorbance at 260 nm. The purity of the isolated RNA was determined by using the 260/280 nm absorption ratio, which was >1.8, as assumed (NanoDrop spectrophotometer, Thermo Scientific, Waltham, MA, USA). The quality and integrity of RNA was also checked in a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). The resulting RNA integrity numbers (RINs) ranged from 8.5 to 10, with a mean of 9.2 (Agilent Technologies, Inc., Santa Clara, CA, USA). Each RNA sample was diluted to a concentration of 50 ng/μL. For the microarray experiments, 100 ng of total RNA was used. The remaining isolated RNA material was used for the RT-qPCR study.