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

Lyons PA, Koukoulaki M, Hatton A, Doggett K, Woffendin HB, Chaudhry AN, Smith KG - BMC Genomics (2007)

Bottom Line: 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.Leukocyte subsets should be prepared for microarray analysis by rapid positive selection.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

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.

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Related in: MedlinePlus

Microarray analysis of purified cell populations from six normal controls. (A) Hierarchical clustering of microarray data generated for five cell populations isolated from six normal controls was performed using expression data from 12,022 genes and clusters samples according to cell lineage. (B) Relative expression levels of CD14, CD16, CD19, CD4 and CD8 mRNA in each of the five cell types.
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Figure 1: Microarray analysis of purified cell populations from six normal controls. (A) Hierarchical clustering of microarray data generated for five cell populations isolated from six normal controls was performed using expression data from 12,022 genes and clusters samples according to cell lineage. (B) Relative expression levels of CD14, CD16, CD19, CD4 and CD8 mRNA in each of the five cell types.

Mentions: To validate the separation protocol, RNA samples extracted from leucocyte subsets from six normal controls were labelled and hybridised to Affymetrix U133 Plus2 GeneChips (Figure 1). Hierarchical clustering of the samples based on expression data from 12,022 genes determined to be present in all replicates of at least one cell type clusters the samples according to cell lineage (Figure 1A). As an additional validation step we examined the expression profiles of a panel of 39 known cell-specific markers, comprised predominantly of CD antigens [see Additional file 1]. Hierarchical clustering of the samples based on the expression data of these 39 genes again clustered the samples according to cell lineage, with the expression pattern of individual CD antigens being as predicted [see Additional file 2]. For example, mRNA for CD74, the invariant chain, is expressed highly in all CD14+ monocyte and CD19+ B cell samples but not in any other cell type [see Additional file 2].


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

Lyons PA, Koukoulaki M, Hatton A, Doggett K, Woffendin HB, Chaudhry AN, Smith KG - BMC Genomics (2007)

Microarray analysis of purified cell populations from six normal controls. (A) Hierarchical clustering of microarray data generated for five cell populations isolated from six normal controls was performed using expression data from 12,022 genes and clusters samples according to cell lineage. (B) Relative expression levels of CD14, CD16, CD19, CD4 and CD8 mRNA in each of the five cell types.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828063&req=5

Figure 1: Microarray analysis of purified cell populations from six normal controls. (A) Hierarchical clustering of microarray data generated for five cell populations isolated from six normal controls was performed using expression data from 12,022 genes and clusters samples according to cell lineage. (B) Relative expression levels of CD14, CD16, CD19, CD4 and CD8 mRNA in each of the five cell types.
Mentions: To validate the separation protocol, RNA samples extracted from leucocyte subsets from six normal controls were labelled and hybridised to Affymetrix U133 Plus2 GeneChips (Figure 1). Hierarchical clustering of the samples based on expression data from 12,022 genes determined to be present in all replicates of at least one cell type clusters the samples according to cell lineage (Figure 1A). As an additional validation step we examined the expression profiles of a panel of 39 known cell-specific markers, comprised predominantly of CD antigens [see Additional file 1]. Hierarchical clustering of the samples based on the expression data of these 39 genes again clustered the samples according to cell lineage, with the expression pattern of individual CD antigens being as predicted [see Additional file 2]. For example, mRNA for CD74, the invariant chain, is expressed highly in all CD14+ monocyte and CD19+ B cell samples but not in any other cell type [see Additional file 2].

Bottom Line: 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.Leukocyte subsets should be prepared for microarray analysis by rapid positive selection.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

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.

Show MeSH
Related in: MedlinePlus