PC from WM patient, isolated using Kappa or Lambda-fluorescein isothiocyanate, CD10-PE, CD38-PerCPCy5.5,CD19-PE-Cy7, CD34-APC and CD45-APC-Cy7.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from purified cell populations using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).
Label
BIOTIN
Label protocol
Labeling were performed according to protocols from Affymetrix. Briefly,100 ng of total RNA was amplified and labeled using the GeneChip two-cycle cDNA synthesis kit and GeneChip IVT labeling kit (Affymetrix Inc., Santa Clara, CA, USA)
Hybridization protocol
Hybridizations were performed according to protocols from Affymetrix. Labeled RNA was hybridized to Human Genome U133A microarray (Affymetrix), after quality checking on GeneChips Test 3 Arrays.
Scan protocol
Washing and scanning were performed using Fluidics Station 400 and GeneChip Scanner (Affymetrix Inc.).
Description
None
Data processing
Expression value for each probe set was calculated using
RMAExpress program that uses RMA (robust multi-array
average) algorithm For a first supervised analysis,
gene-filtering methods were applied following the detection
calls calculated using MAS 5.0 algorithm (Affymetrix). Probe sets
with absent calls as well as those showing minimal variation
across all samples (maximum–minimum log2 variation < 2.5)
were filtered out. In a second, more exhaustive analysis, the
complete set of microarrays without gene filtering was
introduced in a multivariable matrix where each of the six cell
states (NBL, WM-BL, CLL-BL, NPC, WM-PC and MM-PC) were
introduced to perform the differential expression analyses.
