The effect of cell subset isolation method on gene expression in leukocytes

Area of Science:

• Immunology
• Molecular Biology
• Genomics

Background:

• Accurate gene expression analysis relies on pure blood cell subsets.
• Impact of cell isolation methods (positive selection, negative selection, FACS) on gene expression is largely unknown.

Purpose of the Study:

• To compare the effects of positive selection, negative selection, and FACS on gene expression profiles of isolated CD4+ T cells, CD8+ T cells, B cells, and monocytes.
• To determine the optimal method for preserving disease- or treatment-related gene expression signatures.

Main Methods:

• Isolation of CD4+ T cells, CD8+ T cells, B cells, and monocytes from five healthy donors using positive immunomagnetic selection, negative immunomagnetic selection, and FACS.
• Generation of microarray gene expression data.
• Unsupervised clustering and differential gene expression analysis.

Main Results:

• Gene expression profiles were more similar between negative selection and FACS compared to positive selection.
• Contrary to hypothesis, FACS did not upregulate stress response genes.
• Positive and negative selection showed higher expression of stress-response genes compared to FACS.

Conclusions:

• FACS is the recommended method for leukocyte subset isolation for gene expression studies due to high purity and minimal stress response induction.
• Cell isolation method significantly impacts gene expression profiles, necessitating careful selection for accurate downstream analysis.