MIAME checklist for array analysis of A498(HA-pVHL30) vs. A489(neo)
Experiment design
Type of experiment: Comparison of the RCC cell line A498 that is deficient for
pVHL expression with a subclone containing a stably re-introduced HA-tagged
pVHL30 wild-type allele.
Experimental factor: genetic variation (ectopic expression of HA-pVHL30).
Number of hybridizations: 6 (3 replicates each of control (neo) and of HA-pVHL30expressing samples). Single-colour labeling was performed for each sample.
Samples used
A498 cells, established from a clear cell renal cell cancer of a 52 years old male
patient (Giard, D. J. et al. In vitro cultivation of human tumors: establishment of cell
lines derived from a series of solid tumors. J Natl Cancer Inst 51, 1417-23 (1973))
were obtained from ATCC and maintained in DMEM supplemented with 10% fetal
calf serum (FCS). This cell line does not express functional pVHL (Chen, F. et al.
Suppression of growth of renal carcinoma cells by the von Hippel-Lindau tumor
suppressor gene. Cancer Res 55, 4804-7 (1995)). A498 subclones stably transfected
with HA-pVHL30 or control plasmid have been described (Lisztwan, J., Imbert, G.,
Wirbelauer, C., Gstaiger, M. & Krek, W. The von Hippel-Lindau tumor suppressor
protein is a component of an E3 ubiquitin-protein ligase activity. Genes Dev 13, 182233 (1999)) and were maintained in 0.5 mg/ml G418 at 80% cell density.
Sample preparation
Total RNA was prepared using TRIZOL reagent (Life Technologies) and purified on
RNeasy Miniprep columns (Quiagen) according to the manufacturers' instructions
Labeling protocol
10 µg of total RNA was reverse transcribed using the SuperScript Choice system for
cDNA synthesis (Life Technologies) according to the protocol recommended by
Affymetrix. The oligonucleotide used for priming was 5’ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo). Double-stranded
cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed
by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was
performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript
labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up
columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH
8.1, 100 mM KOAc, 30 mM MgOAc.
Hybridization protocol
10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was
controled by use of the GeneChip™ Eukaryotic Hybridization Control Kit
(Affymetrix). Washing and staining was performed in a Fluidics Station 400
(Affymetrix) using the protocol EukGE-WS2v4.
Measurement data, specifications and data processing
Scanning was performed in an Affymetrix GeneChip scanner. Chip analysis was
performed using the Affymetrix Microarray Suite v5 and GeneSpring 4.2.1 (Silicon
Genetics).
A complete list of raw data can be found under the accession number E-MEXP-13 in
the EBI ArrayExpress database (http://www.ebi.ac.uk/arrayexpress).
Changes in gene expression were assessed by looking for concordant changes
between all replicates using a signed Wilcoxon rank test. The “change” p-value
threshold was
< 0.003 for increase and > 0.997 for decrease. After concordance
analysis these values become < 9 x10-6 and > 0.999991 respectively. Any gene whose
detection p-value was > 0.05 in all experimental conditions was discarded from the
analysis as being unreliable data. Cluster analysis was performed using dCHIP
software (www.biostat.harvard.edu/complab/dchip).
Array design
Microarray analysis was performed using HG_U95A version2 GeneChips™
oligonucleotide arrays (Affymetrix 900303) representing approximately 10,000 fulllength genes. Sequences and GenBank accession numbers of all probe sets are
available at the Affymetrix home page http://www.affymetrix.com/index.affx.