Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT ™) and standard density gradient

BMC Immunology

Tomasz I. Michalak³  Werner Kroll²  Sylwia Karwowska¹  Christopher Burgess¹  Danielle P. Ings³  Christopher P. Corkum³ 

[1] Novartis Oncology Companion Diagnostics, Cambridge 02139, MA, USA;Present address: Quidel Corporation, San Diego 92130, CA, USA;Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John’s A1B3V6, NL, Canada

关键词: Affymetrix microarray;    Gene expression analysis;    DNA;    RNA;    Ficoll density gradient;    Immune cell subsets;    BD Vacutainer cell preparation tube;    Peripheral blood mononuclear cells;   

Others  :  1229031
DOI  :  10.1186/s12865-015-0113-0

 received in 2015-05-01, accepted in 2015-08-17,  发布年份 2015

【 摘 要 】

Background

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. 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.

Results

The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 10 ⁶cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 10 ⁶cells/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.

Conclusions

Our 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.

【 授权许可】

   
2015 Corkum et al.

【 图 表 】

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