Bone marrow (BM) CD34+ cells were obtained from PV (n=26), and ET (n=24) patients. CD34+ cells were collected from 5 ml of BM aspirates, all obtained in preservative free heparin. Mononuclear cells were separated over a Ficoll-Paque gradient (Lympholyte; Cederlane Labs) and processed through two sequential steps of immunomagnetic CD34 selection (Miltenyi Biotec, Bergisch Gladbach,Germany,http://www.miltenyibiotec.com). Purity of the isolated CD34+ cell population was evaluated by flow cytometry after labeling with PE-HPCA2 anti-CD34 monoclonal antibody (BD Biosciences) and was always > 95%.
Extracted molecule
total RNA
Extraction protocol
CD34+ cells were purified from BM aspirates and immediately lysed in 700uL of QIAZOL Buffer (Qiagen, Valencia, CA, USA). Total RNA from CD34+ cells was extracted using miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s recommendations for Purification of Total RNA, Including Small RNAs, from Animal Cells. 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 profiling (GEP) and miRNA expression profiling (miEP) were performed on the same RNA preparation. As regards miEP, total RNA (100ng for samples) were labeled using the FlashTag® Biotin HSR kit (Affymetrix).
Hybridization protocol
Regarding miEP, tagged RNA was hybridized to the Affymetrix Genechip miRNA array 2.0 using manufacturer's recommendations (FlashTag™ Biotin HSR RNA Labeling Kit, P/N 703095 Rev. 1). The miRNA arrays were washed and stained by GeneChip Hybridization, Wash and Stain Kit (Affymetrix) with Fluidic Station 450 using fluidic script FS450_003.
Scan protocol
Affymetrix Genechip miRNA arrays 2.0 were scanned using the Affymetrix GCS3000 7G Scanner.
Data processing
Gene and miRNA 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.
