CD34+ cells were isolated from human umbilical cord blood and differentiated into mast cells using rhIL-6 and rh-SCF for 6-8 weeks.
Extracted molecule
total RNA
Extraction protocol
RNA samples were isolated from hCBMCs and CD34- using the RNeasy Mini Kit (Qiagen) including the optional DNase treatment, according to the manufacturer's instructions. Total RNA was quantified using a Nanodrop Spectrophotometer 2000 (Thermo Scientific Inc., Waltham, MA, USA) and the quality confirmed (RIN > 7.0) on an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
biotin
Label protocol
250 ng total RNA was used as input for amplification using Ambion WT expression sense kit (Life Technilogies, Grand Island, NY, USA), Poly A control was added as suggested by WT expression kit manual. cDNA amplification products were fragmented and labeled with biotin using Affymetrix GeneChip WT terminal labeling kit, hybridized to HG Gene 1.0 ST arrays (Affymetrix Inc., Santa Clara, CA, USA) using Affymetrix Hybridization Control Kit, and then washed and stained using Affymetrix Wash and Stain kit. All steps were processed following the manufactures suggestions.
