miRNA profile of WI-38 cells and their progressively transformed derivatives

microRNA array of 4 cell lines: WI-38 primary fibroblasts (“Control”), slow growers (early passage after immortalization, Slow), fast growers (extensive passaging after immortalization) and fast growers transformed by constitutively activated mutant H-RasV12 (“Ras”) WI-38 cells were grown in 37° in MEM supplemented with 10% non-heat-inactivated fetal bovine serum (Sigma), pen-strep, sodium pyruvate, L-glutamine solution (Beit HaEmek). RNA was extracted using Nucleospin miRNA kit (Macherey-Nagel), according to manufacturer’s instructions. microRNA array analysis was done in duplicates, using the miRNA Complete Labeling and Hyb Kit (Agilent, 5190-0456) according to the manufacturer's instructions. Briefly, for each sample 100ng RNA was dephosphorylated, denatured, labeled with Cyanine 3-pCp and purified using Micro Bio-Spin 6 Columns. Hybridization was done for 20h with Agilent SurePrint G3 Unrestricted miRNA 8x60K (Release 19.0) arrays. Arrays were scanned using an Agilent DNA microarray scanner, and analyzed using the AgiMicroRna package in R with the RMA algorithm. The function filterMicroRna was applied with the following parameters: control = TRUE, IsGeneDetected = TRUE, wellaboveNEG = TRUE, limIsGeneDetected = 50 and limNEG = 25.