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Cryopreserved Human B Cells as an Alternative Source for Single Cell mRNA Analysis

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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.

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Abbreviations

CXCR4:

chemokine (CXC family) receptor 4

DMSO:

dimethyl sulfoxide

FACS:

fluo-rescence-activated cell sorting

GAPD:

glyceraldehyde phosphate dehydrogenase

IgVH:

immunoglobulin heavy chain variable region gene

mRNA:

messenger ribonucleic acid

RT-PCR:

reverse transcription-polymerase chain reaction

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Authors and Affiliations

  1. Outpatients Department of Medicine, Charite University Medicine Berlin, Free University and Humboldt University Berlin, Campus Charite Mitte (CCM), Luisenstraße 11-13A, Schumannstr. 20/21, D-10117, Berlin, Germany

    Arne Hansen & Karin Reiter

  2. Institute for Transfusion Medicine (Tissue Bank & Research Unit), Charite University Medicine Berlin, Free University and Humboldt University Berlin, Campus Charite Mitte (CCM), Schumannstr. 20/21, D-10117, Berlin, Germany

    Karin Reiter, Thomas Dörner & Axel Pruss

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  1. Arne Hansen
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  2. Karin Reiter
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  4. Axel Pruss
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Correspondence to Arne Hansen.

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Hansen, A., Reiter, K., Dörner, T. et al. Cryopreserved Human B Cells as an Alternative Source for Single Cell mRNA Analysis. Cell Tissue Banking 6, 299–308 (2005). https://doi.org/10.1007/s10561-005-8348-2

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  • DOI: https://doi.org/10.1007/s10561-005-8348-2

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