Scanning perform with a Agilent 2565C DNA Microarray scanner using default parameters : 100% PMT, 10%PMT (XDR mode), 5 um resolution, at 20°C in low ozone concentration environment.
Agilent Hybridization Protocol (Gene expression Hyb kit Large (ref 5188-5280), Chamber type: Agilent SureHyb Chamber; Quantity of labelled extract: 825ng; duration: 17 hours; volume: 110 ul ; Temperature in °C: 65). Add the following components in clean 1.5 ml tubes: 825 ng linearly amplified cRNA labeled with Cy5 for each tumor, and 825 ng linearly amplified cRNA labeled with Cy3. Conversely, for dye swapped arrays, mix 825 ng linearly amplified cRNA labelled with Cy3 for each tumor, and 825 ng linearly amplified cRNA labeled with Cy5. In each case, add 11 ul 2X Blocking Agent, 2.2 ul of Fragmentation buffer 25X and nuclease free water up to 110 ul. Mix by vortexing. Incubate at 60°C for 30 minutes in dark. Spin briefly, add 110 ul of 2X hybridization buffer. Mix gently. Assembly the Sure-Hyb hybridization chamber from Agilent. Place a backing side with the plastic inner on upper side. Gently add 650 ul of 1x hybridisation solution and cover with the Agilent 4x44k array properly oriented (active surface in contact with liquid). Finish to assembly the chamber and tight the screw. Hybridization carry out for 17 hours at 65°C in a rotating oven (Robbins Scientific, Mountain View, CA) at 4 rpm. The arrays are disassembled at room temperature in SSPE Wash 1 Buffer (ref 5188-5327), then wash 1 minute in a glass dish (Wheathon) at room temperature in Wash 1 Buffer, then 1 minute in SSPE Wash 2 Buffer (ref 5188-5327) at 37°C
(500 ng, Amplification=RNA polymerases). Agilent oligo Cy5 or Cy3 probes labeling protocol. Kit used for probe labeling: Agilent Low RNA Input Linear Amplification kit (ref 5188-5340) adapted for small amount of total RNA (500 ng total RNA per reaction). In 1.5 ml tubes add 500 ng total RNA from sample. Add 1.2 ul T7 promoter primer, 2 ul Spike In dilution 1/3200 (ref 5188-5279), use Spike In A to label Cy3 target and Spike In B to label Cy5 target, then add nuclease free water (Invitrogen ref:10977-015) to bring the volume up to 11.5 ul. Denature by incubating at 65°C for 10 minutes. Place the reactions on ice and incubate 5 min. Spin briefly. Prepare a Reverse Transcription master mix, adding for one reaction 4 ul First strand Buffer 5X, 2 ul DTT 0.1M, 1 ul dNTP 10 mM mix, 0.5 ul Random Hexamer, 1 ul MMLV Reverse Transcriptase (200 U/ul), 0.5 ul RNAse out (40 U/ul). Master mix is prepared in batch for all the samples included in the study (vol per one reaction multiplied by number of samples. In each reaction tube containing denaturated RNA and T7 promoter primer in a volume of 11.5 ul, add 9 ul of Reverse Transcriptase master mix, and mix by gently pipetting. Incubate at 40°C in a circulating water bath for 2 hours. Move samples to 65 °C for 15 minutes to inactivate MMLV RT, and incubate on ice for 5 minutes. Spin briefly. Prepare a in vitro transcription master mix, adding for one reaction : 20.1 ul Nuclease free water, 20 ul Transcription buffer 4X, 6 ul DTT 0.1 M, 8 ul NTP mix, 0.5 ul RNAse out, 0.6 ul Inorganic Pyrophosphatase, 0.8 ul T7 RNA Polymerase. Transcription master mix is prepared in batch for all the samples included in the study: (vol per one reaction multiplied by number of samples). Master mix is splited in two aliquots, one for Cy5 and one for Cy3. Add 1.6 ul (multiplied by reactions number) CTP-Cy5 25 mM (Perkin Elmer ref NEL 581) or 2.4 ul CTP-Cy3 (multiplied by reactions number) (Perkin Elmer, ref NEL 582) to a total volume of 60 ul/ reaction. To each RT reaction, add 60 ul Transcription master mix. Incubate at 40°C for 2 hours. Add 20 ul nuclease free water and freeze at –20°C. Labeled probes are purified using Qiagen Rneasy mini kit and protocol provided by Agilent. For each probe, add 350 ul RLT buffer, and 250 ul ethanol 100 °. Mix by gently vortexing. Apply 700 ul on Rneasy columns and spin at 13,000 g for 30 s at 4°C. Discard flow-through. Wash twice with RPE buffer. Dry the column and elute in 60 ul nuclease free water. Measure concentration and Cy5/3 incorporation using a Nanodrop spectrophotometer. Adjust concentration at 100 ng/ul. Freeze at –20°C until hybridization.
The cohort for microarray analysis corresponded to patients treated in three medical centers (Centre Hospitalier Universitaire Lyon-Sud, Centre Hospitalier Universitaire Lille, AP-HM Marseille, France) and included successively between September 1997 and May 2008 (n = 108). Patients were selected according to the following criteria: primary tumour with no evidence of distant metastasis at the time of diagnosis. The tumours were frozen immediately upon excision by surgeon and supported by the biobank sites
RNA extraction :Tumour biopsies were snap-frozen in liquid nitrogen upon surgical removal and stored in liquid nitrogen until RNA extraction. The tumours were included in PEG and cut frozen. RNAs were prepared from 25 to 30 sections of 25 µm thickness. One pathologist (PPB) reviewed tumour sections prior to analysis. To be finally included, the specimen should hold at least 70% of tumour cells. Total RNAs were extracted using TRI Reagent (Sigma, St Louis MO). To remove any genomic DNA contamination, total RNAs were treated with RNAse-free DNAse I and purified using RNeasy micro columns (Qiagen, Hilden, Germany). RNAs quality was verified using an Agilent Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA) and we retained RNA samples with RNA Integrity Number more than 7.5 for microarray analysis and more than 6.5 for RT QPCR analysis. Concentration of all RNAs is adjusted at 100 ng/ul, and verified with a second measurement on Nanodrop spectrophotometer