Gene expression in primary human bone marrow plasma cells sorted according to CD19 expression

To characterize human bone marrow plasma cells that express or lack CD19 on a molecular level, we compared the global gene expression of primary CD38high/CD138+ plasma cells with or without CD19 expression. Primary human bone marrow plasma cells (PC) from immunologically healthy donors were enriched according to CD138 expression by magnetic cell separation. From the enriched fraction, CD19-positive and CD19-negative PC were isolated by flow cytometric cell sorting, according to the phenotype CD38high/CD138+/CD20-/CD3-/CD14-/DAPI-/CD19+ and CD38high/CD138+/CD20-/CD3-/CD14-/DAPI-/CD19-, respectively, using a BD Aria or DIVA cell sorter (Becton Dickinson). Dead cells were excluded using DAPI. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 100 ng total RNA using Message AmpII Biotin (Ambion, USA). To obtain sufficient cRNA, the Affymetrix two-cycle amplification protocol was used. Fifteen micrograms of fragmented cRNA of each sample were hybridized to a total of 8 HG-U133 Plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality-checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.