Fluorescence-intensities were filtered using following criteria: For miRNAs consistent fluorescence-intensities for all of the triplicate spots on each array. For both miRNAs and mRNAs spots with net median fluorescence intensities less than twice median background fluorescence intensity were excluded from further analysis.Single channel analysis was achieved using log2 net fluorescence-intensities (median values, with background subtracted) normalized by scaling from 0 to 1. False discovery rates (FDR) computed according to Benjamini & Hochberg (Hochberg and Benjamini, 1990) were used to correct selection of genes for false positives (P<0.05). Normalization between arrays was carried out using Z-score normalization. Statistical analysis of microarray data was carried out on data derived from triplicate slides derived from three separate transfections. Results from batches of slides were combined into a single data-file for statistical analysis. The ANOVA facility of the SPOTFIRE program was used to isolate differentially expressed miRNAs or mRNAs (P<0.05 or 0.01).
Hybridization of labelled samples of RNA was carried out at 41C for 18 h in a SlideBooster 400 Hybridization Station (Advalytix, Munich, Germany). Microarray analysis was carried out in technical and biological triplicates.
The microarrays were scanned in a Packard Bioscience Scanarray Lite microarray scanner (Perkin-Elmer, Waltham, MA, USA). The Cy3 and Cy5 fluorescence signals were quantified by using the ScanArrayExpress v.3.0 software (Perkin-Elmer, Waltham, MA, USA). The fluorescence intensities (contained in .txt-files) were analysed using the SPOTFIRE v. 9.0 DecisionSite for Microarray Analysis software (TIBCO Software, Palo Alto, CA, USA).
Total RNA was extracted 72 hours after transfection from E10 cells transfected with miR-363-5p mimics, miR-20b or scrambled control, using the RNeasy Mini Kit as described by the manufacturer’s (Qiagen, Hilden, Germany). The RNA samples were treated with the TURBO DNA-free kit (Applied Biosystems, CA, USA) to remove contaminating genomic DNA. RNA-fractions enriched with respect to miRNAs, were isolated according to the manufacturers protocol from E10 cells transfected with miRNA mimics or scrambled control using the mirPremier microRNA isolation kit (Sigma-Aldrich, St. Louis, MO, USA). Concentrations of solutions of purified RNA were assayed using the Nanodrop ND-1000 spectrophotometer. RNA fractions exhibiting a ratio of OD260/OD280 and OD260/OD230 of at least 1.8 and 2.0, respectively, were used for further analysis. The quality and composition of solutions of RNA was assessed using the Agilent-Bioanalyzer (Agilent, Palo Alto, CA, USA).
Human squamous carcinoma cell line (clone E10 (PE/CA-PJ49) was obtained from the European Collection of Cell Cultures (ECACC). The cells were grown in Iscove’s Modified Dulbecco’s Medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10 % (v/v) FBS, 2 mM L-glutamine, 1 % (v/v) penicillin, 1 % (v/v) streptomycin and 0.25 ug/ml amphotericin B (all reagents purchased from Lonza, Basel, Switzerland). The cells were split twice weekly at 80% confluence. Cells (E10) were incubated in IMDM medium with 5 % FBS and transfected with INTERFERin (Polyplus-Transfection, Illkirch, France) according to the manufacturers protocol using 20 nM of Allstar scrambled control (Qiagen, Hilden, Germany), miR-20b or miR-363-5p (miR-363*) mimic. All miRNA mimics were purchased from GenePharma, Shanghai, China.
Human deoxyoligonucleotide microarrays for miRNA (OneArray Microarray v2) or mRNA (OneArray Microarray v4 and v5) were purchased from Phalanx Biotech Group (Belmont, CA, USA). The miRNA arrays contain 100% of Sanger miRBase v15 miRNA content, i.e., 1087 unique miRNA probes, and probes for mRNA arrays were derived from RefSeq and Ensembl sequences. All miRNA probes were printed in triplicates. MiRNA-enriched fractions were labelled using Kreatech ULS labeling of miRNA (Kreatech, Amsterdam, Netherlands). Each slide was hybridized with samples containing 1 ug of RNA. mRNA arrays were hybridized using cDNA prepared from fraction containing total RNA isolated using RNeasy mini kit (Qiagen GmbH, Hilden, Germany). cDNA synthesis and labelling of cDNAs with Cy3 or Cy5 were carried out using the Genisphere 900 labelling kit (Genisphere LLC, Hatfield, PA, USA).