5 protocols
The arrays were scanned using the Affymetrix Gene Chip Scanner 3000 7G. The scanned images were processed using the AGCC (Affymetrix GeneChip Command Console) Software and the CEL files were imported into Partek Genomics Suite software (Partek, Inc. MO, USA).
Affymetrix GeneChip Exon 1.0 arrays
All pediatric samples start with B and all adult samples start with V. Hence Pr 3, 4, 70 and 80 are children and Pr 1, 2, 50 and 60 are adults. When possible 5 subpopulations from each person was sorted. In successive maturation these are: ProB, PreBI, PreBII large, PreBII small and Immature B cells.
Total RNA was extracted and purified from each precursor B cell subset using the miRNeasy Mini Kit (Qiagen, Hilden Germany) and Phase Lock Gel Heavy (5 PRIME GmbH, Hamburg, Germany). RNA was quantified with a NanoDrop ND-1000 Spectrophotometer (Saveen Werner, Malmo, Sweden), or if too low concentration, by NanoDrop ND-3300 Fluorospectrometer (Saveen Werner, Malmo, Sweden) using the RiboGreen method (Molecular Probes, Invitrogen detection technologies, Eugene, OR, USA). Quality was assessed with Agilent 2100 Bioanalyzer using either the Agilent RNA 6000 Nano Kit or Agilent RNA 6000 Pico Kit (Agilent Technologies, Palo Alto, CA, USA) depending on sample concentration. The samples concentrations ranged from 3.9 to 149.5 ng/ul and from 6.4 to 9.9 (mean 8.4, n = 39) for Bioanalyzer RIN indicating high RNA purity and integrity.
Bone marrow aspirates from 4 healthy children and four adults