Microarray images are analysed by using Feature extraction software version 10.7.3.1 from Agilent technologies and Agilent normalization protocol GE2_107_Sep09 with 028004_D_F_20111107 as design. Default settings are used. Raw data files from the Agilent Feature Extraction software 10.7.3.1 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 incorporation bias.
The hybridization were performed using the following Agilent Hybridization Protocol (Chamber type: Agilent SureHyb Chamber; Quantity of labelled extract: 1650 ng per labelling; duration: 17 hours; volume: 100 uL per array; Temperature in _C: 65). The steps were the following: Add 1650 ng of linearly amplified cRNA labeled Cy3 in clean 1.5 ml tubes. Add 11 uL of Blocking Agent 10X, 2.2 uL of Fragmentation buffer 25X and nuclease free water up to 55 uL. Mix by vortexing. Incubate at 60_C for 30 minutes in dark. Spin briefly; add 55 uL of 2X hi-hybridization buffer. Mix gently. Assembly the Sure-Hyb hybridization chamber from Agilent. Place a backing slide with the plastic inner on upper side. Gently add 100 uL of 1x hybridization solution and cover with the Agilent 8x60k (AMADID 028004) GE Human 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 eventually dried using a nitrogen gun, and scanned by using an Agilent G2565CDNA microarray scanner.
Gene expression assay were performed using the Agilent oligo Cy3 probes labelling protocol. The kit used for probe labelling (Agilent Low Input Quick Amp Labeling Kit, one-color (5190-2305)) was adapted for small amount of total RNA (100 ng total RNA per reaction). Samples were processed as follow: In 1.5 mL tubes add 100 ng total RNA from sample. Add 0.8 uL T7 promoter primer, 2uL spike-in A for Cy3 labelling and add nuclease free water (Invitrogen ref: 10977-015) to bring the volume to 5.3 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 2 uL of first strand Buffer 5X, 1 uL of DTT 0.1M, 0.5 uL of dNTP 10 mM mix, 1.2 uL of Affinity Script Block Mix. Master mix is prepared in batch for all the samples included in the study (volume per one reaction multiplied by number of samples). In each reaction tube containing denaturated RNA and T7 promoter primer in a volume of 5.3 uL, add 4.7 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 enzymes, and incubate on ice for 5 min. Spin briefly. Prepare an in vitro transcription master mix, adding for one reaction: 0.75 uL of nuclease free water, 3.2 uL of transcription buffer 5X, 0.6 uL of DTT 0.1 M, 1 uL of NTP mix, 0.24 of CTP-Cy3, 0.21 uL of T7 RNA Polymerase Blend. Transcription master mix is prepared in batch for all the samples included in the study (volume per one reaction multiplied by number of samples). To each RT reaction, add 6 uL of transcription master mix. Incubate at 40_C for 2 hours. Add 84 uL of nuclease free water and freeze at -20_C. Labelled probes are purified using Qiagen Rneasy mini kit and protocol provided by Agilent. For each probe, add 350 uL of RLT buffer, and 250 uL of 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 of nuclease free water. Measure concentration and Cy5/Cy3 incorporation using a Nanodrop spectrophotometer. Adjust concentration at 100 ng/uL. Freeze at -20_C until hybridization.
Scanning perform with a Agilent G2505C DNA Microarray scanner using default parameters : 20 bits mode, 3 um resolution, at 20_C in low ozone (controlled) concentration environment.
Isolation of CD34+ cells :CD34+ cells were obtained, after informed consent in agreement with our Institute Ethic Committee (Assistance Publique des Hôpitaux de Paris) and in accordance with the Declaration of Helsinki, either from leukapheresis samples after mobilization performed on patients or from umbilical cord blood. CD34+ cells were isolated by a positive selection using an immunomagnetic cell sorting system (AutoMacs; Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in serum-free medium in the presence of recombinant human thrombopoietin (TPO; 10 ng/mL; Kirin Brewery, Tokyo, Japan).
Total RNAs were extracted using the TriReagent method (TriReagent, Euromedex, Strasbourg, France). For each sample, the following sequence was performed as quickly as possible: Pellet cells were placed in 1.6 mL of TriReagent. For separation of the aqueous phase and isopropanol precipitation, cells homogenates (1.6 mL) were added by 320 uL of chloroform and incubated at room temperature for 15 min and then centrifuged at 12,000 g for 15 min at 4_C. The upper aqueous phase (about 800 uL) containing RNA was collected. RNAs were then precipitated from the aqueous phase by addition of isopropanol (one volume isopropanol for one volume of aqueous solution) 10 min at room temperature followed by a centrifugation at 12,000 g 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 at -20_C. To assess the quality of RNAs, 1 uL was used for determination of concentration using a Nanodrop spectrophotometer (http://www.nanodrop.com). The 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. The ratio of 18s/28s ribosomal RNA ranges from 0 to 2.3, and the RNA Integrity Number (RIN) from 2.3 to 9.4. Usually, RNA showing a RIN lower than 7 with an 18s/28s ratio below 1.8 are discarded from study. Since these samples are rare and difficult to obtain, they underwent the hybridization procedure (GE single color assay as well as miRNA assay). Concentrations of all RNAs were adjusted at 80 ng/uL, and verified with a second measurement on a Nanodrop spectrophotometer. These RNAs were used for GE and microRNA assays.
Liquid serum-free medium and purification of the CD41+ cell population : CD34+ were cultured in serum-free medium in the presence of TPO (10 ng/mL; Kirin Brewery, Tokyo, Japan). Ingredients used to prepare the serum-free medium were as previously described 28. To purify the CD41+ population, cells in suspension culture were collected at day 6-7, stained with an anti–CD41a-PE monoclonal antibody (MoAb) (PharMingen, San Diego, CA) and purified by cell sorting (Influx, Becton Dickinson, le Pont de Claix, France). The purified CD41+ cell population was subsequently grown in the same serum-free liquid medium with or without TPO and in presence or absence of S78454 for an additional 24 h. When stated, CD41+ cells were starved overnight from cytokine. Then CD41+ cells were stimulated for different times with TPO with or without S78454; cells were subsequently recovered in dry pellet for subsequent analyses.