Microarray images are analysed by using Feature extraction software version 220.127.116.11 from Agilent technologies and Agilent normalization protocol GE2-v5_95_Feb07 with 014850_D_F_20060807 as design. Default settings are used. Raw data files from the Agilent Feature Extraction software 18.104.22.168 for image analysis were imported into Resolver(TM) system (version 22.214.171.124) for gene expression data analyses from Rosetta Inpharmatics. Then, combined experiments were generated to obtain average values from the dye swap experiments in order to avoid dye effect bias.
Scanning perform with a Agilent 2565B DNA Microarray scanner using default parameters : 100%PMT, 10%PMT (XDR mode), 5 um resolution, at 20 C in low ozone concentration environment.
(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 65C 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 40C 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 40C for 2 hours. Add 20 ul nuclease free water and freeze at –20C. 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 4C. 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 –20C until hybridisation.
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 60C 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 44k 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 65C 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 37C. Slides are scanned by using an Agilent 2565 AB DNA microarray scanner.
Total RNA is isolated from using Rneasy micro elute kit (Qiagen, Courtaboeuf, France) according to manufacturer instructions. Briefly Cells is mixed with 350 ul RLT buffer with Beta Mercaptoethanol 1%. Then add 350 ul ethanol 70% and mix thoroughly by vortexing. The mixture is transferred to a RNA-binding spin cup and centrifuged at 8,000 g for 20 seconds. The spin cup is washed with 350 ul 1x Low-Salt Wash Buffer, incubated with RNase-Free DNase I at 37C for 15 minutes, washed with 350 ul 1x High-Salt Wash Buffer, then washed with 500 ul 1x Low-Salt Wash Buffer and finally with 500ul ethanol 80%. Total RNA is eluted by addition of 15 ul Nuclease free water to the RNA-binding spin cup and centrifugation at 20,000 g for 1 minute. 1 ul of the eluted RNA is used for determination of concentration using Nanodrop spectrophotometer (www.nanodrop.com). Quality of RNA-preparations is assessed using Lab-on-a-chip Bioanalyser 2000 technology (Agilent technologies), based on the 28S/18S ribosomal RNAs ratio. All samples included in this study displayed a ratio of ribosomal RNAs between 1.5 and 2. Concentration of all RNAs is adjusted at 100 ng/ul, and verified with a second measurement on Nanodrop spectrophotometer.