BioProject

High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. More...

High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. In this study, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays. Overall design: PBMC from 3 healthy donors were isolated using Ficoll-Paque gradient or BD Vacutainer CPT methods. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods. RNA was extracted from total PBMC isolated by both protocols and from their subsets, followed by analysis of gene expression using Affymetrix microarrays. Gene expression was compared between Ficoll and CPT samples for a given cell type. Less...