Abstract
BackgroundHigh 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. Due to the lack of the relevant data published, 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 evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.ResultsThe mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.ConclusionsOur findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells’ handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0113-0) contains supplementary material, which is available to authorized users.
Highlights
High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology
About 45 ml of blood was obtained from each donor using Becton Dickinson (BD) Vacutainer tubes containing acid-citrate-dextrose anticoagulant, solution A (ACD-A; BD) from which Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque gradient centrifugation
As described in the Methods, a red blood cells (RBC) lysis step was included after purification of PBMC by both Ficoll and cell preparation tube (CPT) protocols to minimize the impact of potentially contaminating RBC- and reticulocyte-derived RNA on downstream RNA and gene expression analyses
Summary
High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. The PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles. Several factors, including sample collection, storage and transportation, manipulation, and extraction of nucleic acids and their preservation methods can affect the integrity of test material [15,16,17,18]. Careful consideration must be given when designing protocols utilizing the above technologies
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