Document Detail


Cryopreserved human B cells as an alternative source for single cell mRNA analysis.
MedLine Citation:
PMID:  16308769     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Arne Hansen; Karin Reiter; Thomas Dörner; Axel Pruss
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Cell and tissue banking     Volume:  6     ISSN:  1389-9333     ISO Abbreviation:  Cell Tissue Bank     Publication Date:  2005  
Date Detail:
Created Date:  2005-11-25     Completed Date:  2006-02-22     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100965121     Medline TA:  Cell Tissue Bank     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  299-308     Citation Subset:  IM    
Affiliation:
Outpatients Department of Medicine, Charite University Medicine Berlin, Germany, ahansen@charite.de
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MeSH Terms
Descriptor/Qualifier:
Antigens, Surface
B-Lymphocytes / metabolism*
Cryopreservation*
Flow Cytometry
Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
Humans
Immunoglobulin gamma-Chains / genetics
RNA, Messenger / genetics*,  isolation & purification*
Receptors, CXCR4 / genetics
Reverse Transcriptase Polymerase Chain Reaction
Chemical
Reg. No./Substance:
0/Antigens, Surface; 0/Immunoglobulin gamma-Chains; 0/RNA, Messenger; 0/Receptors, CXCR4; EC 1.2.1.-/Glyceraldehyde-3-Phosphate Dehydrogenases

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