2.3. One color microarray microRNA expression analysis

One hundred nanograms of total RNA from cells at different experimental conditions were treated following the miRNA microarray protocol (Agilent Technologies, Placerville, CA). Briefly, RNA was dephosphorylated and denatured, followed by a ligation and labeling step. Samples were hybridized to Human miRNA Microarray 8 × 60K glass arrays from the Sanger miRBase database release 16 (2006 human miRNAs represented, Agilent Technologies). After hybridization, slides were washed following the Agilent procedure and scanned with the dual-laser Agilent Scanner version C (G2505C, Agilent Technologies). Images were analyzed using the Feature Extraction software v10.7. Raw data elaboration was carried out with Bioconductor (www.bioconductor.org),³² using R statistical language. The LIMMA package was then used to identify differentially expressed miRNAs between the different experimental conditions. The empirical Bayes method was used to compute a moderated t-statistics.³³ miRNAs with a log base two-fold change (logFC) greater than +0.58 or lower than −0.58 were considered as differentially expressed.