cell type: MPD disease: Primary mielofibrosis (PMF) tissue: peripheral blood (PB) v617f jak2: pos mplw51: neg asxl1 mut: pos srsf2 mut: pos ezh2 mut: neg at least one mutation: pos
Biomaterial provider
Vannucchi Lab
Growth protocol
CD34+ cells were purified from 30–50 ml of Peripheral Blood (PB) collected from primary mielofibrosis (PMF) patients. Mononuclear cells were separated over a Ficoll-Paque gradient (Lymphoprep; Nycomed Pharma, Asker, Norway, http://www.nycomed.com) and processed through two sequential steps of immunomagnetic CD34 selection (Miltenyi Biotec, Bergisch Gladbach,Germany,http://www.miltenyibiotec.com).
Extracted molecule
total RNA
Extraction protocol
CD34+ cells were purified from Peripheral blood of PMF patients and immediately lysed in 700uL of QIAZOL Buffer (Qiagen, Valencia, CA, USA). Total RNA from CD34+ cells was extracted using miRNeasy Mini 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
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.
Hybridization protocol
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.
Scan protocol
GeneChip were finally scanned using GeneAtlas Scanner.
Description
Peripheral Blood CD34+ cells from PMF patient
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.
