2.5. RNA Isolation and microRNA Assay

RNA was isolated utilizing standard phenol-chloroform extraction procedures as previously described [35]. RNA quality was determined by Nanodrop using the 8000 Spectrophotometer (Thermo Scientific, Wilmington, NC, USA) and RNA integrity and presence of the small RNA fraction was determined using the Bioanalyzer 2100 capillary electrophoresis system (Agilent, Santa Clara, CA, USA). Sixty nanograms of total RNA was reverse transcribed using the human Megaplex Primer Pools A and B and the TaqMan miR reverse transcription kit (Applied Biosystems, Foster City, CA, USA) [35]. Each sample was pre-amplified for 12 cycles using human pool A and B Taqman^® Megaplex™ PreAmp Primers and PreAmp Master Mix (Applied Biosystems) and the preamplification reactions diluted, combined with TaqMan ^® Gene-Expression Master Mix (Applied Biosystems) divided into eight aliquots and each aliquot was added to one of the eight sample ports of the TaqMan^® Array A or B (v2.0), respectively. The TaqMan^® Array Human miR Card Set v2.0 (Thermo Fisher Scientific, Waltham, MA, USA) enables detection of 667 human miRs, 3 miR endogenous reference controls, and 1 miR assay not related to human as a negative control. Table S1 includes relevant miR platform and sequence information. The real-time PCR reactions were run according to the manufacturer’s instructions. RealTime Statminer Software (Integromics, Philadelphia, PA, USA) was used to analyze the data. The global geometric mean of all expressed miR assays was used to normalize the data. Significantly altered miRs were determined based on the most stringent criteria with a p-value cutoff of 1E-05 was used to determine statistically significant miRs.