miRNA Profiling of Cancer Cell Lines

miRNA profiling of 67NR and 4T1.2 cell lines was conducted using Exiqon miRCURY LNA microarrays (Qiagen, Australia). Briefly, total cell RNA (2 μg) isolated using Trizol (ThermoFisher) was labelled with 1 µL of Cy3 or Cy5 (500 ng/μL) fluorescent dinucleotide (Dharmacon, UK) in a buffer containing 0.1 mM ATP, 50 nM HEPES (pH 7.8), 2.5 mM DTT, 20 mM MgCl₂, 10 mg/mL BSA, and 10% DMSO with 20 units RNA ligase (New England Biolabs) for 2 h at 4 °C. RNA was precipitated with 0.3 M sodium acetate (pH 5.2), 20 µg glycogen and 75% ethanol, washed with 70% ethanol, mixed and co-hybridised to the array in duplicate dye-swapping reactions at 60 °C overnight. Following washing, slides were analysed with a GenePix 4000B Scanner (Molecular Devices, USA) and differential expression analysed in Limma [71 (link)], using tools for linear modelling and the empirical Bayes statistic calculations.