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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

Delaying separation leads to significant changes in gene expression especially in cells of the myeloid lineage. Expression profiles were obtained from RNA samples extracted from cells separated immediately following venesection compared to those separated after a four hour delay on ice. Box plots (A) show the change in gene expression between 0 and 4 hours for independent experiments (I – III) and combined self versus self hybridisation data. The Venn diagrams (B) show the number and overlap between genes showing statistically significant differential expression (as defined in the materials and methods).
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Figure 4: Delaying separation leads to significant changes in gene expression especially in cells of the myeloid lineage. Expression profiles were obtained from RNA samples extracted from cells separated immediately following venesection compared to those separated after a four hour delay on ice. Box plots (A) show the change in gene expression between 0 and 4 hours for independent experiments (I – III) and combined self versus self hybridisation data. The Venn diagrams (B) show the number and overlap between genes showing statistically significant differential expression (as defined in the materials and methods).

Mentions: Storing blood prior to separation has no noticeable effect on the separation process. The final purities for all four cell subsets were not statistically different irrespective of whether the blood was stored prior to separation or not (data not shown). However, storage has a clear effect on RNA levels (Figure 4).


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)

Delaying separation leads to significant changes in gene expression especially in cells of the myeloid lineage. Expression profiles were obtained from RNA samples extracted from cells separated immediately following venesection compared to those separated after a four hour delay on ice. Box plots (A) show the change in gene expression between 0 and 4 hours for independent experiments (I – III) and combined self versus self hybridisation data. The Venn diagrams (B) show the number and overlap between genes showing statistically significant differential expression (as defined in the materials and methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Delaying separation leads to significant changes in gene expression especially in cells of the myeloid lineage. Expression profiles were obtained from RNA samples extracted from cells separated immediately following venesection compared to those separated after a four hour delay on ice. Box plots (A) show the change in gene expression between 0 and 4 hours for independent experiments (I – III) and combined self versus self hybridisation data. The Venn diagrams (B) show the number and overlap between genes showing statistically significant differential expression (as defined in the materials and methods).
Mentions: Storing blood prior to separation has no noticeable effect on the separation process. The final purities for all four cell subsets were not statistically different irrespective of whether the blood was stored prior to separation or not (data not shown). However, storage has a clear effect on RNA levels (Figure 4).

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