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Published on: October 25, 2018
Microarray analysis of human leucocyte subsets: the advantages of positive selection and rapid purification
Paul A Lyons1, Maria Koukoulaki, Alexander Hatton
1Department 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
Insights
To accurately study immune-related diseases, researchers must profile purified leukocyte subsets from blood. Rapid positive selection is the optimal method for isolating these cells for gene expression analysis.
Area of Science:
- Immunology
- Molecular Biology
- Genomics
Background:
- Expression profiling of peripheral blood cells is crucial for understanding immune-related diseases.
- Previous studies using total peripheral blood mononuclear cells often reflected cell abundance differences, not disease-specific transcriptional changes.
- Profiling purified leukocyte subsets is necessary to identify cell-specific gene expression alterations.
Purpose of the Study:
- To establish a reliable method for isolating specific leukocyte subsets from peripheral blood for gene expression analysis.
- To compare the effectiveness of positive and negative selection methods for cell purification.
- To assess the impact of cell storage on gene expression profiles.
Main Methods:
- Sequential rounds of positive selection were employed to isolate CD4 T cells, CD8 T cells, CD19 B cells, CD14 monocytes, and CD16 neutrophils.
- Gene expression was analyzed using microarray analysis.
- Comparisons were made between cells isolated via positive selection and negative selection, and the effect of pre-separation cell storage was evaluated.
Main Results:
- Positive selection effectively isolated major leukocyte subsets (T cells, B cells, monocytes, neutrophils) from a single blood sample.
- No significant gene expression changes were consistently observed due to positive selection.
- Negative selection yielded inferior results, primarily due to reduced cell purity.
- Storing cells before separation caused substantial gene expression changes, particularly in myeloid cells.
Conclusions:
- Rapid positive selection is the recommended method for preparing leukocyte subsets for microarray analysis.
- This method ensures high purity and reliable gene expression data for immune-related disease research.
- Avoiding pre-separation cell storage is critical for accurate transcriptional profiling.
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.
