diagnosis: Stage 4 neuroblastoma age at diagnosis: > 1 year survivor: dead with disease within 36 months from diagnosis tissue: at least 80% of neuroblasts
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from frozen tumors by PerfectPureTM RNA Tissue Kit (5Prime, Hamburg, Germany), including RNase-free DNase I treatment (Ambion, TX, USA). Total and small RNAs were quantified and quality control was assessed by RNA 6000 Nano® and Small RNA® assays, respectively, on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
We performed miRNA expression profiling starting from 100 µg of total RNA and following the miRNA Microarray System protocol v. 1.5 (Agilent Technologies). In dephosphorylation and ligation steps, 17 Units of Calf Intestine Alkaline Phosphatase (Takara Bio Inc., Shiga, Japan) and 20 Units of T4 RNA ligase (Takara Bio Inc.) have been used, respectively.
Hybridization protocol
We followed the miRNA Microarray System protocol v. 1.5 (Agilent Technologies).
Scan protocol
Slides were scanned by Agilent G2565BA scanner according to the miRNA Microarray System protocol v. 1.5 (Agilent Technologies).
Description
Images were processed by Feature Extraction software v. 9.5.3.1 (Agilent Technologies).
Data processing
Tab-delimited text file were analyzed in R v. 2.7.2 software environment (www.r-project.org) using the limma package v. 2.14.16 (16) of Bioconductor (www.bioconductor.org). Only spots with signal minus background flagged as positive and significant were used in the following analysis as ‘detected’ spot. Probes with less than 50% of detected spots across all arrays were removed from the analysis. Similarly, arrays with a number of detected spots smaller than 50% of all spots on the array were removed. Background corrected intensities of replicated spots on each array were averaged. Data were then log2-transformed and normalized for between-array comparison using quantile normalization (Bolstad, B. M., Irizarry R. A., Astrand, M., and Speed, T. P. (2003), A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics 19, 185-193).
