Flash tag biotin hsr

The microarray analysis was performed on the GeneAtlas™ Microarray System (Affymetrix). Total RNA from each samplewas labeled with biotin in accordance to Affymetrix Flash Tag™ Biotin HSR (Ref. 901,913, Affymetrix) kit instructions. The labeled molecules were hybridized onto Affymetrix miRNA 4.1 Array Strip (Affymetrix) using the GeneChip GeneAtlas™ Hybridization and Stain Module (Ref. 902,135) reagents (Affymetrix). After hybridization and washing, the arrays were scored on the Imaging Station of the GeneAtlas™ Microarray System. Array data of the samples have been deposited in the Array Express database at EMBL-EBI (www.ebi.ac.uk/arraexpress) under the accession no. E-MTAB-7060.

Total RNA from paraffin tissues was isolated using the RNeasy FFPE kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The total RNA was quantified using NanoDrop One (Thermofisher Scientific, Inc., Waltham, MA, USA). For miRNA expression analysis, a standard protocol was followed. Briefly, 250 ng of total RNA from each sample was labeled using the FlashTag™ Biotin RNA Labeling kit (Affymetrix^®; Thermo Fisher Scientific, Inc.), and the labeled RNA was hybridized with the GeneChip miRNA 4.0 Array (Affymetrix^®; Thermo Fisher Scientific, Inc.). The miRNA microarray chips were washed twice with 1X PBST (0.02% Tween) buffer and stained with FlashTag™ Biotin HSR (Affymetrix; Thermo Fisher Scientific, Inc.) for 5 min at 35 °C. CEL files were generated after digitizing the image. The fluorescence intensity values in CEL format were pre-processed and normalized by quartiles using Expression Console™ v1.4.1.46 software (Affymetrix; Thermo Fisher Scientific, Inc.). Transcriptomic Analysis Console™ v4.0 software (Affymetrix; Thermo Fisher Scientific, Inc.) was used for expression analysis and heatmap generation, where the CHP files generated after normalization were loaded. Differentially expressed miRNAs were identified by ±1.5-fold change and eBayes p-values of < 0.05 between each sample group.