Total RNA was extracted from biopsies with TRIzol reagent, purified and DNAse treated using the SV Total RNA Isolation System (Promega, Leiden, The Netherlands). RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands) with 6000 Nano Chips according to the manufacturer’s instructions. RNA was judged as suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RIN (RNA integrity number) above 8.0.
Label
biotin
Label protocol
Total RNA (500 ng) extracted from biopsies was labelled using the Ambion MessageAmp II biotin enhanced single-round amplification kit (cat. no. 1791).
Hybridization protocol
The labelled RNA was hybridised to a GeneChip Hu133 Plus2 array (Affymetrix, Santa Clara, CA), washed, and stained. Detailed methods for the labelling and subsequent hybridisations to the arrays are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix.
Scan protocol
The GeneChip Hu133 Plus2 array was scanned on an Affymetrix GeneChip 3000 7G scanner. Detailed methods are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix
Description
none
Data processing
Packages from the Bioconductor project, integrated in an in-house developed on-line management and analysis database for multiplatform microarray experiments, were used for analysing the scanned Affymetrix arrays (Gentleman et al., 2004; Gavai et al, submitted).
