Exiqon Microarrays and Data Analysis
Total RNA purification, including miRNAs, was performed using the miRNeasy kit (QIAGEN) and samples were stored immediately at −80°C. RNA quantification and integrity was assessed using Nanodrop and Agilent 2100 Bioanalyzer. Only samples with a RNA integrity number (RIN) >8,0 were taken for analysis. A total of 500 ng RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA Array version 5th Generation (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE version 16.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 9.0 software (BioDiscovery, Inc., USA). The quantified signals were background corrected (Normexp with offset value 10  and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Differential expression of miRNAs between groups was performed using a t-test one-tail after which a p value <0.05 was considered statistically significant. A total of 52 miRs were used to generate a heatmap where red and green colors indicate high and low expression respectively. A two-way supervised clustering analysis was performed using Pearson’s correlations and Ward’s criteria as a linkage rule. C27IM cell line was hybridized twice with correlation = 0,92. Microarrays data have been deposited in the Curie database at http://microarrays.curie.fr/, login username and password are available upon request. Full paper
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