Isolation and Profiling of Total RNA

Total RNA was prepared from 10 to 50 mg of frozen tissue on the days of treatment, preincubated with RNAlater-ICE (Ambion, Austin, TX, USA) using the miRNeasy Mini kit (Qiagen, Hilden, Germany) and TissueRuptor (Qiagen) following the manufacturers' instructions. The RNA quality was checked by the RNA 6000 Nanoassay on the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples were divided into two identical aliquots for independent labelling and hybridisation. Total RNA for each sample (0.5 mg) was amplified and biotinylated using the Illumina TotalPrep RNA Amplification Kit (Ambion) according to manufacturers' standard procedure. This consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using a reverse transcriptase (RT). The quality and quantity of cRNA in the samples was checked with an Agilent Bioanalyzer 2100 (Agilent Technologies), samples were diluted to 150 ng ml⁻¹ and hybridised to Illumina HT-12 BeadChips in duplicate (Illumina, San Diego, CA, USA). This Illumina platform was previously validated by qPCR in a breast cancer-derived xenograft study (Sims et al., 2012 (link)).