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


CONCLUSIONS
Leukocyte subsets should be prepared for microarray analysis by rapid positive selection.
Lyons
Paul A
PA
Department of Medicine, and Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2XY, UK. pal34@cam.ac.uk
Koukoulaki
Maria
M
Hatton
Alexander
A
Doggett
Karen
K
Woffendin
Hayley B
HB
Chaudhry
Afzal N
AN
Smith
Kenneth G C
KG
D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D016131:Lymphocyte Subsets / Q000166:cytology*; D008863:Microspheres; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*
D012313:RNA