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

Corkum CP, Ings DP, Burgess C, Karwowska S, Kroll W, Michalak TI - BMC Immunol. (2015)

Bottom Line: Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. c.corkum@mun.ca.

ABSTRACT

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(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) 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.

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.

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Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2
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Fig8: Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2

Mentions: Previous reports have demonstrated that experimental handling and manipulation, including the method of tissue collection and preparation, can adversely affect gene expression in primary cells and tissues, which can interfere with the biological interpretation of results [15–18]. To compare gene expression between cell subsets from PBMC prepared by Ficoll and CPT isolation procedures, gene expression profiles were generated on the Affymetrix Human Genome U133 Plus 2.0 Array. We first performed PCA using all genes to visualize the global distribution of gene expression. PCA is a mathematical technique used to determine the key sources of variation in a multidimensional dataset by reducing the dimensionality of the dataset by finding new variables, termed principle components (PC). Using these principle components, each sample can be characterized by a few values which can be plotted, thus allowing a visual assessment of differences and similarities between samples [38]. PCA has been widely used in the analysis and visualization of microarray data [41, 42]. In the PC#1 vs PC#2 plot shown in Fig. 8, it is clearly evident that the predominant variation in gene expression between samples was attributable to cell type. Samples of the same cell type formed distinct clusters away from other cell types in the PC#1 vs PC#2 plot, which accounted for 19.3 % and 12.1 % of the total variance, respectively (Fig. 8). Given their similar cellular onotology, both T cell subset samples, CD8+ and CD4+, were grouped together more closely than the other immune cell types, indicating a close relation in terms of gene expression. Interestingly, samples processed with either Ficoll or CPT, within a given cell type, were also found closely grouped. Further analyses of the signal scatter plots comparing the intensities of gene expression between total PBMC and individual immune cell subsets purified from these PBMC obtained by either Ficoll or CPT gave similar results as above. The pattern of gene expression also was very similar between Ficoll and CPT-derived PBMC and their immune cell subsets obtained from each donor, with Pearson’s correlation coefficients greater than 0.96 for all comparisons (Fig. 9). Taken together, these results showed that in regard to a given cell type there was little or no difference in gene expression between the two PBMC isolation methods.Fig. 8


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

Corkum CP, Ings DP, Burgess C, Karwowska S, Kroll W, Michalak TI - BMC Immunol. (2015)

Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4549105&req=5

Fig8: Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2
Mentions: Previous reports have demonstrated that experimental handling and manipulation, including the method of tissue collection and preparation, can adversely affect gene expression in primary cells and tissues, which can interfere with the biological interpretation of results [15–18]. To compare gene expression between cell subsets from PBMC prepared by Ficoll and CPT isolation procedures, gene expression profiles were generated on the Affymetrix Human Genome U133 Plus 2.0 Array. We first performed PCA using all genes to visualize the global distribution of gene expression. PCA is a mathematical technique used to determine the key sources of variation in a multidimensional dataset by reducing the dimensionality of the dataset by finding new variables, termed principle components (PC). Using these principle components, each sample can be characterized by a few values which can be plotted, thus allowing a visual assessment of differences and similarities between samples [38]. PCA has been widely used in the analysis and visualization of microarray data [41, 42]. In the PC#1 vs PC#2 plot shown in Fig. 8, it is clearly evident that the predominant variation in gene expression between samples was attributable to cell type. Samples of the same cell type formed distinct clusters away from other cell types in the PC#1 vs PC#2 plot, which accounted for 19.3 % and 12.1 % of the total variance, respectively (Fig. 8). Given their similar cellular onotology, both T cell subset samples, CD8+ and CD4+, were grouped together more closely than the other immune cell types, indicating a close relation in terms of gene expression. Interestingly, samples processed with either Ficoll or CPT, within a given cell type, were also found closely grouped. Further analyses of the signal scatter plots comparing the intensities of gene expression between total PBMC and individual immune cell subsets purified from these PBMC obtained by either Ficoll or CPT gave similar results as above. The pattern of gene expression also was very similar between Ficoll and CPT-derived PBMC and their immune cell subsets obtained from each donor, with Pearson’s correlation coefficients greater than 0.96 for all comparisons (Fig. 9). Taken together, these results showed that in regard to a given cell type there was little or no difference in gene expression between the two PBMC isolation methods.Fig. 8

Bottom Line: Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, NL, A1B3V6, Canada. c.corkum@mun.ca.

ABSTRACT

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(6) cells/ml, 93.3%) were compatible to those obtained with Ficoll (1.34 × 10(6) 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.

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.

Show MeSH