feature extractoin protocol
Microarray images were analysed by using Feature Extraction software version ( 10.5.1.1) from Agilent technologies. Defaults settings were used.
array scanning protocol
Scanning was performed with a Agilent G2565C DNA Microarray scanner using defaults parameters (100░ PMT, 5 um resolution, at 20░C in low ozone concentration environment. Microarray images were analysed by using Feature Extraction software version (10.5.1.1) from Agilent technologies. Defaults settings were used.
hybridizationnucleic acid hybridization to array protocol
Agilent Hybridization Protocol (Chamber type: Agilent SureHyb Chamber; Quantity of labelled extract: 825 ng per labelling ; duration: 17 hours; volume: 100 ul per array; Temperature in ░C: 65). Add the following components in clean 1.5 ml tubes: 825 ng linearly amplified cRNA labeled with Cy5 for sample 1, and 825 ng linearly amplified cRNA labeled with Cy3 for sample 2. Conversely, for dye swapped arrays, mix 825 ng linearly amplified cRNA labelled with Cy3 for sample1, and 825 ng linearly amplified cRNA labeled with Cy5 for sample 2. In each case, add 50 ul Bocking Agent 10X, 10 ul of Fragmentation buffer 25X and nuclease free water up to 250 ul. Mix by vortexing. Incubate at 60░C for 30 minutes in dark. Spin briefly, add 250 ul of 2X hi-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 490 ul of 1x hybridisation solution and cover with the Agilent 244k exon array properly oriented (active surface in contact with liquid). Finish to assembly the chamber and tight the screw. Hybridization was carried out for 17 hours at 65░C in a rotating oven (Robbins Scientific, Mountain View, CA) at 10 rpm. The arrays were disassembled at room temperature in wash 1 buffer (Agilent), then washed 1 minute in a glass dish (Wheathon) at room temperature in wash 1 buffer, then 1 minute in wash 2 buffer at 37░C and then 1 minute in acetonitril. Slides were dried using a nitrogen gun, and scanned by using an Agilent G2565 C DNA microarray scanner.
(500 ng, Amplification=RNA polymerases). Agilent oligo Cy5 or Cy3 probes labelling protocol. Kit used for probe labelling: Agilent Fluorescent Low input Linear Amplification kit (G2554A) 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, 2ul Spike-in A for Cy3 labelling or B for Cy5 labelling and add nuclease free water (Invitrogen ref:10977-015) to bring the volume to 11 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), 1 ul nuclease free water. 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 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 3 hours. Add 20 ul nuclease free water and freeze at û20░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/Cy3 incorporation using a Nanodrop spectrophotometer. Adjust concentration at 100 ng/ul. Freeze at û20░C until hybridisation.
nucleic acid extraction protocol
Total RNAs were extracted using the TriReagent method (TriReagent, Euromedex, Strasbourg, France). 1) Tissue lysis/homogenization. For each sample, the following sequences were performed as quickly as possible: Cuts of the samples were placed in 1ml TriReagent and grinded by using a tissue ruptor. Then 600╡l of TriReagent were added. 2) Separation of the aqueous phase and isopropanol precipitation. Tissue homogenates (1,6 ml) were added by 320 ul of chloroform and incubated at room temperature for 15 min and then centrifuged at 12á000 g for 15 min at 4░C. The upper aqueous phase (about 800 ul) containing RNA was collected. RNA was then precipitated from the aqueous phase by addition of isopropanol (1 volume isopropanol for one volume aqueous solution) 10 min at room temperature and centrifugation at 12 000g for 10 min at 4░C. Pellets were washed twice with ethanol 75 % (7 500 g, 5 min, 4░C), resuspended in 100 ml sterile RNAse-free water, and frozen in û20░C. 4) Quality control. One microliter was used for determination of concentration using a Nanodrop spectrophotometer (www.nanodrop.com). Quality of RNA-preparations was further 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 1.8 and 2. Concentration of all RNAs was adjusted at 100 ng/ul, and verified with a second measurement on a Nanodrop spectrophotometer.