Fresh surgical tissues were immediately snap-frozen in liquid nitrogen, stored at −80 °C and microdissected prior to RNA extraction and only specimen with at least 80% of tumor cells were included in the study. Total cellular RNA was extracted using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA) according to the anufacturer’s instructions. The RNA was reverse transcribed to single-stranded cDNA using the High Capacity Kit (Applied Biosystems, Foster City, CA, USA).
Label
Cy3
Label protocol
The microRNA library was constructed using 100 ng of total RNA and investigated with the Agilent Human microRNA Microarray Kit (v3)” (G4470C, Agilent Technologies, Santa Clara, CA, USA) to determine the expression profile of microRNAs according to manufacturer’s instructions. This array contains 866 human and 89 human viral microRNA sequences. Each miRNA species is printed 20 times with replicate probes on the array.
Hybridization protocol
The labeled RNA was purified (MicroBioSpin 6, Bio-Rad, 732-6221), hybridized (overnight for 20 hours at 55°C and 20 rpm) and washed (Agilent, 5188-5327) before scanning.
Scan protocol
Microarray slides were scanned at 535 nm with 5 μm/pixel resolution using a DNA Microarray Scanner with SureScan High-Resolution Technology (Agilent Technologies) and the fluorescent intensities were determined by the Feature Extraction Software Version 11.5 (Agilent Technologies), to obtain Processed Signals (resulting from all intra-array pre-processing, including background corrections) corresponding to the transcript levels.
Data processing
The spot images were processed with Feature Extraction Software v10.7.3.1 (Agilent Technologies). Each miRNA microarray was analyzed with respect to signal intensity, background signal, as well as internal control blade and spike in controls. A box-plot was generated in order to determine the distribution of signals from each spot on each microarray as well as sample integrity and quality. An empirical Bayesian approach was used to determine differential expression and the false discovery rate (FDR) was used to control the false-positive rate. Normalization was performed by the 75% quartile method.
