Cells were homogenized in TRIzol reagent (Invitrogen, Karlsruhe, Germany) using the FastPrep FP120 cell disruptor (Qbiogene, Inc, Carlsbad, CA, USA).Total RNA was isolated following the TRIzol protocol (Invitrogen) and RNA integrity was assessed using the 2100 Bioanalyzer (Agilent, Waldbronn, Germany). Only samples with an RNA Integrity Number >7.5 were taken for further analysis.
Label
Cy3
Label protocol
Labeling was performed according to Agilent's recommentdations. In brief, 1µg total of tumor RNA was linearily amplified and labeled with Cy3 using Agilent's one-color Quick Amp Labeling Kit following the instructions of the protocol.
Hybridization protocol
Hybridization was performed following the manufacturer's protocol. In brief, 1650 ng of Cy3-labeled cRNA was hybridized on 4x44K custom microarrays using Agilent's High-RPM Gene Expression Hyb Kit. Hybridization was performed for 17 hours at 65°C in a rotating hyb oven at 10 rpm according the company's recommendations.
Scan protocol
After washing and scanning, resulting TIFF-images were processed using Agilent's Feature Extraction software Version 9.5.1.
Description
Tumour cDNA NB_4_T
Data processing
Global gene expression analyses was performed using customized single channel Agilent arrays (4x 44k “NB-custom arrays”, GPL 16876) as described (Kocak et al., Cell Death Dis, 2013). Microarrays were analysed using R (r-project.org; version 3.1.0) and the following packages: arrayQualityMetrics (version 3.20.0), GEOquery (version 2.30.1), Biobase (version 2.22.0), marray (version 1.42.0) and limma (version 3.18.13). A gene-wise linear model (limma) was used to identify significantly differentially expressed genes between primary and relapse NB samples.
