7 protocols
Raw data were pre-processed to filter out those probes with more than 40% of measurements classed as not available. The filter on the coverage of the probes was done to reduce biases in the normalisation step. Pre-processed data were normalised using Quantile method (Bolstad et al., 2003). ComBat algorithm was used to adjust batch effects due to the execution of the experiments in different laboratories and in different times (Walker et al., 2008).
The arrays were washed and scanned with a laser confocal scanner (G2565BA, Agilent Technologies) according to the manufacturer's instructions. miRNA microarrays underwent standard post hybridisation processing and the intensities of fluorescence were calculated by Feature Extraction software version 11 (Agilent Technologies).
Labeled samples were hybridized on G4470B human miRNA Microarray kit (Agilent Technologies), according to the manufacturer's instructions.
tumour samples from a cohort of 183 patients staged according to the FIGO criteria as stage I EOC were gathered from a frozen tissue bank containing 1300 samples that were collected between September, 1992, and March, 2005, and available at the Department of Oncology, Mario Negri Institute, Milan, Italy. Tumour tissue had been collected from patients who underwent surgery for EOC at the Obstetrics and Gynecology Department, San Gerardo Hospital, Monza, Italy, The collection and use of tumour samples was approved by the local scientific ethical committees and written consent was obtained from the patients.
100 ng of total RNA enriched in miRNA fraction was Cy3-labelled and hybridised with a miRNA labelling and hybridisation kit according to the manufacturer's instructions (Agilent Technologies).
HOSE cells were prepared as described previously (Auersperg et al., 1994), and stabilized for 1-6 passages at confluence before use.
Frozen samples (30 mg) were homogenised in an Ultraturrax at 4°C, and total RNA enriched in miRNAs fraction was purified using a mirVana isolation kit according to the manufacturer's instructions (Ambion-ABI, Milan, Italy). RNA was measured by Nanodrop, and the presence of small RNAs was checked with a 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). Aliquots were stored at −80°C until use.