All RNA was prepared on the same platform by the CsCl-cushion, as described (Chirgwin, 1979, Biochemistry, 18, 5294-5299.). The quality and the quantity of RNA samples were evaluated by Agilent 2100 Bioanalyser RNA LabChip kit (Agilent Technologies, Palo Alto, CA, USA).
Label
33P
Label protocol
Between 0.4 and one µg of total RNA was used as template to generate cDNA bearing T7 promoter, then antisense RNA (aRNA) was generated by in vitro transcription using MEGAscript technology according to the Ambion protocol (Ambion Inc., Austin TX). An aliquot of 2µg of labelled aRNA was then primed with random hexaprimers and reverse transcribed with a mix of cold dNTPs and [a-33P]dCTP.
Hybridization protocol
Labelled cDNAs were then hybridized in 0.3 ml hybridization mix in scintillation vials for 48 h at 68° C. After hybridization filters were washed twice in 0.1x SSC, 0.1% SDS at 68° C for 90 min.
Scan protocol
DNA microarrays were scanned at 25-µm resolution using a Fuji BAS 5000 image plate system (Raytest, Paris, France). The hybridization signals were quantified using ArrayGauge software v.1.3 (Fuji, Ltd, Tokyo, Japan).
Description
228 patients (included in clinical trial PACS01) + 24 patients (included based on the same inclusion criteria as those used for PACS01 trial).
Data processing
Data were background noise subtracted (negative controls ± 6 SD), then normalized by original global intensity of hybridization. After expression data correction for the amount of PCR product spotted onto the membrane (testing probe data), data were log2-transformed and finally standardized. Only genes well measured for 70% of patients were retained.
