To induce lineage-specific differentiation, growth factors (R&D Systems, Wiesbaden-Nor-denstadt, Germany, were added as follows: for erythropoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), IL-3 (10 ng/ml), EPO (10 U/ml); for granulopoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), IL-3 (10 ng/ml), G-CSF, and GM-CSF (each, 10 ng/ml); for megakaryopoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), TPO (20 ng/ml). All growth factors were added at the beginning of culture. The cells were cultured in a final volume of 2 ml, in separate wells for each of the conditions and each of the time points. The cells were inspected daily for 11 days and harvested on days 0, and 11.
Growth protocol
CD34+ PBCs were cultured in X-VIVO10 (BioWhittaker, Walkersville, MD) supplemented with 1% human serum albumin (HSA). At least 1 x 106 CD34+ cells were assayed in 6-well plates and incubated at 37°C and 5% CO2 in a fully humidified atmosphere in air.
Extracted molecule
total RNA
Extraction protocol
Total RNA enriched with microRNA was extracted using RNAeasy mini kit (Qiagen, Valencia, CA) following the manufacturer’s directions. The concentration of the isolated RNA was determined using the Nanodrop ND-100 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Quality and integrity of the total RNA isolated was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Biotin
Label protocol
Total RNA containing low molecular weight RNA was labeled using the Flashtag RNA labeling kit (Genisphere, Hatfield, PA, USA) according to the manufacturer's instructions. Briefly, for each sample, 2 µg total RNA were subjected a tailing reaction (2.5 mM MnCl2, ATP, Poly A Polymerase - incubation for 15 minutes at 37°) followed by ligation of the biotinylated signal molecule to the target RNA sample (1× Flash Tag ligation mix biotin, T4 DNA ligase - incubation for 30 minutes at RT) and adding of stop solution.
Hybridization protocol
Each sample was hybridized to a GeneChip® miRNA Array (Affymetrix, Santa Clara, CA, USA) at 48°C and 60 rpm for 16 hours then washed and stained on Fluidics Station 450 and scanned on a GeneChip® Scanner 3000 7G (Affymetrix). The image data were analyzed with the miRNA QC Tool software for quality control (www.affymetrix.com/products_services/arrays/specific/mi_rna.affx#1_4).
Scan protocol
The raw data were preprocessing by Affymetrix miRNA QC Tool (Version 1.1.1.0) using robust multiarray analysis algrorithm for background correction, quantile normalization and median polish summarization. 4,592 unique mature human miRNAs probes were selected for hierarchical clustering analysis. The differential expression miRNA between cell groups were selected by moderated t-statistics with Benjamini-Hochberg multiple testing correction implemented in R bioconductor limma package.
Data processing
RMA was used to normalize the data. The differential expression miRNA between cell groups were selected by moderated t-statistics with Benjamini-Hochberg multiple testing correction implemented in R bioconductor limma package. Differentially expressed miRNA were selected with statistical filters of log 2 FC and FC greater than 2.0.
