Human CD34+ cells were transfected by using the 4D-Nucleofector™ System (Lonza) as previously reported (Norfo R et al., Blood, 2014). Briefly, starting from the day after CD34+ cell purification, each sample was electroporated twice, once every 24 hours, with mirVana miR-34a-5p mimic or mirVana miRNA mimic Negative Control #1 (Neg-mimic). For each electroporation, 4×10^5 CD34+ cells were resuspended in 100 µL of P3 Primary Cell Solution (Lonza), containing 3 µg of miRNA, and pulsed with the program DS112. After each Nucleofection cycle, cells were maintained in culture in 24-well plates at 5 x 10^5/mL in serum-free medium SYN-H (ABCell-Bio, Paris, France) supplemented with SCF (50 ng/ml), Flt3-ligand (Flt3L) (50 ng/ml), TPO (20ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from Miltenyi Biotec).
Growth protocol
Human CD34+ cells were purified from umbilical cord blood (CB) samples, collected after normal deliveries, according to the institutional guidelines for discarded material. Mononuclear cells were isolated by Ficoll-Hypaque (Lympholyte; Cederlane Labs) gradient separation, washed twice with phosphate-buffered saline, and then CD34+ cells were purified by immunomagnetic sorting (EasySep Human CD34 Positive Selection kit, StemCell Technologies Inc.). The purity of CD34+ cells, assessed by flow cytometry, was always >95%. After immunomagnetic separation, CD34+ cells were seeded in serum-free medium SYN-H (ABCell-Bio, Paris, France) supplemented with SCF (50 ng/ml), Flt3-ligand (Flt3L) (50 ng/ml), TPO (20ng/ml), IL-6 (10 ng/ml) and IL-3 (10 ng/ml) (all from Miltenyi Biotec).
Extracted molecule
total RNA
Extraction protocol
CD34+ cells were lysed in 700uL of QIAZOL Buffer (Qiagen, Valencia, CA, USA) after 24h from the last nucleofection. Total RNA from CD34+ cells was extracted using miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s recommendations. 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. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA.
Label
biotin
Label protocol
Gene expression profile (GEP) was performed on total RNA derived from 3 independent experiments. Briefly, biotinylated aRNA were prepared according to the standard GeneAtlas 3’IVT Express Kit protocol from 100ng of total RNA (P/N 702833 Rev. 4, Affymetrix). Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the aRNA concentration/quality as well as to optimize the aRNA fragmentation. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to evaluate the aRNA sample concentration.
Hybridization protocol
Fragmented cRNA was hybridized for 16h at 45C on Affymetrix HG-U219 array strips in GeneAtlas Hybridization Station. GeneChips were washed and stained using GeneAtlas Hybridization, Wash, and Stain Kit for 3’IVT Arrays, according to the standard protocol supplied by Affymetrix (GeneAtlas 3’ IVT Express Kit, P/N 702833 Rev. 4).
Scan protocol
Affymetrix HG-U219 array strips were scanned by using GeneAtlas Scanner.
Description
GEP of NegCTR mimic CD34+ HPCs at 24 hours post-Nucleofection, third donor
Data processing
Gene expression data were imported into Partek Genomics Suite 6.6 (Partek, St Louis, Mo) as CEL files using default parameters. Raw data were preprocessed, including background correction, normalization, and summarization using robust multiarray average (RMA) analysis.
