supplier: Vannucchi Sex: M condition: myeloproliferative neoplasm (MPN) disease: ET jak2v617f: pos mpl-mutated: neg calr-mutated: neg gene mutation: V617F tissue: Bone marrow cell marker: CD34+
Growth protocol
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 GEP, biotinylated cRNA 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 cRNA concentration/quality as well as to optimize the cRNA fragmentation. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to evaluate the cRNA sample concentration.
Hybridization protocol
Regarding GEP, fragmented cRNA was hybridized for 16 hr 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 finally scanned using GeneAtlas 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.
