6 protocols
normalization data transformation protocol
RNA microarray underwent standard post hybridization processing and the intensities of fluorescence were calculated by features extraction software, version 11 (Agilent Technologies). Raw microarray data then were pre-processed and normalized using the Robust Multichip Average algorithm (RMA), keeping only probes detected in at least 60% of the samples (flagged as IsGeneDetected by Agilent Feature Extraction).
array scanning protocol
Arrays were washed and scanned with laser confocal scanner (G2565BA) according to manufacturer's instructions.
nucleic acid hybridization to array protocol
Labeled RNA was hybridized on the commercially available G13 human miRNA microarray kit (Agilent Technologies), according to the manufacturer's instructions.
nucleic acid labeling protocol
Array experiments were performed using standardized procedure: 100 ng were Cy3-labelled according to manufacturer's instructions (Agilent Technologies, Palo Alto Ca US).
nucleic acid extraction protocol
RNA enriched in miRNAs fraction was purified from the paraffin-embedded slides through robotic workstation (QIAcube, Qiagen) by using miRNeasy FFPE Kit isolation system following manufacturer's protocols (Qiagen). Total RNA concentration and proteins contamination were determined by Nanodrop spectrophotometer (Nanodrop Technologies, Ambion).
growth protocol
Paraffin-embedded (FFPE) material of the surgical specimen or of the diagnostic biopsy of the primary colorectal tumor was used. For each histological sample, a representative hematoxylin and eosin-stained section was reviewed by two independent pathologists. The neoplastic and stromal areas were selected and separately scraped, from corresponding unstained slides.