CD34+ hematopoietic progenitors purified from cord blood (CB). Human CD34+ cells were purified from umbilical cord blood samples, collected after normal deliveries, according to the institutional guidelines for discarded material. Mononuclear cells were isolated by Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, Oslo, Norway) gradient separation, washed twice with PBS, and then CD34+ cells separated using magnetic cell sorting procedure (EasySep Human CD34+ positive selection kit, StemCell Techonologies Inc.; Vancouver, Canada). CD34+ cell purity assessed by flow cytometry were always >95%.
Treatment protocol
Nucleofection of CD34+ stem/progenitor cells was performed according to the optimized protocol provided by the manufacturer, with several modifications (Amaxa Biosystem, Cologne, Germany; www.amaxa.com). 24 hours prior to electroporation the primary CD34+ hematopoietic cells were seeded in 24-well plates at a density of 5x105/ml in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO, Grand Island, NY, USA) cointaining 20% HS (Bio-Whittaker, Walkersville, MD, USA), SCF (50 ng/ml), Flt3-ligand (Flt3-l) (50 ng/ml), TPO (20ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from R&D Systems, Minneapolis, MN, USA). CD34+ cells were subjected to three cycle of nucleofection every 24h. Briefly, for each electroporation 5x105 cells were gently resuspended in 100 µl of Human CD34+ Nucleofector SolutionTM (Amaxa Biosystem), mixed with 5 µg of a mixture of 4 siRNAs targeting CD34 mRNA (SMARTpool, Dharmacon, Lafayette, CO, USA) and pulsed with the program U-01. As a control, in a separate sample, a non-targeting siRNA (siCONTROL Non-Targeting Pool, Dharmacon) was used. Immediately after nucleofection cells were transferred into pre-warmed fresh medium in 24-well plates. Cells were analyzed 24-48 hours after third nucleofection for both viability and CD34 antigen expression.
Growth protocol
To achieve an optimal expansion and differentiation of the primary CD34+ hematopoietic stem/progenitor cells, a serum-free liquid culture was performed by seeding CD34+ cells in 12-well plates at a density of 1x105/ml (2 ml/well) in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO), containing 20% BIT (BIT serum substitute: Bovine serum albumin, Insulin and Transferrin, StemCell Techonologies Inc.), SCF (50 ng/ml), Flt3-ligand (Flt3-l) (50 ng/ml), IL-6 (10 ng/ml) and TPO (10 ng/ml) (all from R&D Systems).
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA was extracted from 0.5x105 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. RNAs originating from 3 different experiments were pooled in order to obtain at least 2 μg per sample.
Label
biotin
Label protocol
One-cycle target labeling assays was performed using Affymetrix standard protocols (Affymetrix, Santa Clara,CA). Briefly, biotin-labeled target synthesis was performed, starting from 2 µg of total cellular RNA, according to the protocol supplied by the manufacturer (Affymetrix, Santa Clara, CA). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation.
Hybridization protocol
The fragmented cRNA was then hybridized to an identical lot of Affymetrix Human HG-U133A GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v4 protocol, using antibody-mediated signal amplification.
Scan protocol
GeneChip were finally scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
Description
Synthesis of biotinylated cRNA targets, arrays hybridization (Mouse430_2 GeneChip, Affymetrix, Santa Clara,CA), staining and scanning were performed using Affymetrix standard protocols starting from 2μg of total RNA.
Data processing
Scaling to TGT value=100. The GeneChip-Operating-Software (GCOS) absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples.
