The clinico-pathological features of the patients have been previously reported. Updated follow-up information was obtained from cancer registry files and approval for the study was granted by the Local Ethical Board. SACs were diagnosed on the basis of criteria proposed by Mäkinen et al., and hmMSI-Hs according to prior established criteria (mucinous, signet-ring cell, and medullary carcinoma, tumor infiltrating and peritumoral lymphocytes, “Crohn-like” inflammatory response, poor differentiation, tumor heterogeneity, and “pushing” tumor border) and later cofirm using MSI-H testing (MSI Analysis System, version 1.2 provided by Promega (Madison, USA)).
Extracted molecule
total RNA
Extraction protocol
A volume of approximately 10mm3 was extracted from each frozen tissue using the disposable sterile biopsy punch Acupunch 2mm (Acuderm Inc, Lauderdale, FL, USA). RNA was extracted following the manufacturer´s instructions (Qiagen, Hilden, Germany). Briefly, tissue was disrupted and homogenized in 700µl of Qiazol (Qiagen ref:1023537) using a Tissueruptor by Qiagen for 20 seconds. The homogenate was incubated at room temperature for 5 minutes. After adding 140µl of chloroform and centrifuging at 12,000xg for 15 minutes at 4ºC, 350µl of the aqueous phase was subjected to automatic total RNA extraction using the Qiacube equipment and the miRNeasy Mini Kit (ref:217004), both provided by Qiagen.
Label
biotin
Label protocol
Total RNA was quantified by spectrometry (NanoDrop ND1000, NanoDrop Technologies, Wilminton, Delaware USA) and quality confirmed by RNA 6000 Nano Bioanalyzer (Agilent Technologies, Palo Alto, California USA) assay. Using NanoDrop measurements, an approximately 50 ng/ul dilution was prepared from each total RNA sample. All steps of the procedure were performed according to the Affymetrix standardized protocol for miRNA 3.0 arrays.
Hybridization protocol
All steps of the procedure were performed according to the Affymetrix standardized protocol for miRNA 3.0 arrays.
Scan protocol
All steps of the procedure were performed according to the Affymetrix standardized protocol for miRNA 3.0 arrays.
Description
miRNA
Data processing
The Affymetrix raw data were normalized using the robust multichip average algorithm from ‘oligo’ Bioconductor package in R version 3.0.1. Normalized miRNA expression data were evaluated using Bioconductor LIMMA differential expression analysis.
