title: Cryopreserved human B cells as an alternative source for single cell mRNA analysis.
abstract: Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.
authors.affiliation: Outpatients Department of Medicine, Charite University Medicine Berlin, Germany, ahansen@charite.de
authors.forename: Arne
authors.initials: A
authors.lastname: Hansen
authors.forename: Karin
authors.initials: K
authors.lastname: Reiter
authors.forename: Thomas
authors.initials: T
authors.lastname: Dörner
authors.forename: Axel
authors.initials: A
authors.lastname: Pruss
mesh_terms: D000954:Antigens, Surface; D001402:B-Lymphocytes / Q000378:metabolism*; D015925:Cryopreservation; D005434:Flow Cytometry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007142:Immunoglobulin gamma-Chains / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification*; D019718:Receptors, CXCR4 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000954:Antigens, Surface; D007142:Immunoglobulin gamma-Chains; D012333:RNA, Messenger; D019718:Receptors, CXCR4; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases