Repository logo
 

Microarray analysis of human leucocyte subsets: the advantages of positive selection and rapid purification.


Change log

Authors

Lyons, Paul A 
Koukoulaki, Maria 
Hatton, Alexander 
Doggett, Karen 
Woffendin, Hayley B 

Abstract

BACKGROUND: For expression profiling to have a practical impact in the management of immune-related disease it is essential that it can be applied to peripheral blood cells. Early studies have used total peripheral blood mononuclear cells, and as a consequence the majority of the disease-related signatures identified have simply reflected differences in the relative abundance of individual cell types between patients and controls. To identify cell-specific changes in transcription it would be necessary to profile purified leucocyte subsets. RESULTS: We have used sequential rounds of positive selection to isolate CD4 and CD8 T cells, CD19 B cells, CD14 monocytes and CD16 neutrophils for microarray analysis from a single blood sample. We compared gene expression in cells isolated in parallel using either positive or negative selection and demonstrate that there are no significant consistent changes due to positive selection, and that the far inferior results obtained by negative selection are largely due to reduced purity. Finally, we demonstrate that storing cells prior to separation leads to profound changes in expression, predominantly in cells of the myeloid lineage. CONCLUSION: Leukocyte subsets should be prepared for microarray analysis by rapid positive selection.

Description

Keywords

Cell Separation, Flow Cytometry, Gene Expression Profiling, Humans, Lymphocyte Subsets, Microspheres, Oligonucleotide Array Sequence Analysis, RNA

Journal Title

BMC Genomics

Conference Name

Journal ISSN

1471-2164
1471-2164

Volume Title

Publisher

Springer Science and Business Media LLC
Sponsorship
Medical Research Council (G0400929)