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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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Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and (b) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences (P < 0.05) in the amount of DNA recovered and in its quality between the two methods of PBMC isolation. Error bars indicate standard error of the mean
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Fig7: Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and (b) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences (P < 0.05) in the amount of DNA recovered and in its quality between the two methods of PBMC isolation. Error bars indicate standard error of the mean

Mentions: Since the yields of viable immune cells from PBMC isolated by Ficoll and CPT were similar, it was expected that this would also hold true when comparing the amounts of RNA and DNA extracted from these cells. As indicated, RNA and DNA were extracted from both total PBMC and individual immune cell subsets isolated from these PBMC. To compare yields, the amount of RNA and DNA was normalized to the number of cells used for extraction. The mean amount of RNA attained from total CPT-PBMC was 0.90 pg/cell, which was similar to 0.86 pg/cell for Ficoll-PBMC (P = 0.871) (Fig. 6a). The average yields of RNA from B cells derived from Ficoll- and CPT-PBMC were 0.73 pg/cell and 0.65 pg/cell, respectively (P = 0.507); CD8+ T lymphocytes, 0.66 pg/cell and 0.50 pg/cell, respectively (P = 0.365); monocytes, 1.43 pg/cell and 1.28 pg/cell, respectively (P = 0.187), and CD4+ T lymphocytes, 0.77 pg/cell and 0.57 pg/cell, respectively (P = 0.266) (Fig. 6a). Similar to RNA yields, there were no significant differences in the yields of DNA (Fig. 7a). Thus, the average amount of DNA extracted from Ficoll-PBMC, and CD19+, CD8+, CD14+ and CD4+ cells derived from these PBMC were 3.76, 5.13, 4.60, 4.01 and 4.42 pg/cell, respectively, compared to 3.74, 5.92, 5.35, 4.03, and 5.71 pg/cell for the same cells derived by CPT isolation protocol (P = 0.977, 0.142, 0.227, 0.955, and 0.141, respectively). Overall, the results indicated that the PBMC isolation method did not affect the number of separated immune cell subsets or the amount of nucleic acids extracted.Fig. 6


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)

Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and (b) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences (P < 0.05) in the amount of DNA recovered and in its quality between the two methods of PBMC isolation. Error bars indicate standard error of the mean
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and (b) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences (P < 0.05) in the amount of DNA recovered and in its quality between the two methods of PBMC isolation. Error bars indicate standard error of the mean
Mentions: Since the yields of viable immune cells from PBMC isolated by Ficoll and CPT were similar, it was expected that this would also hold true when comparing the amounts of RNA and DNA extracted from these cells. As indicated, RNA and DNA were extracted from both total PBMC and individual immune cell subsets isolated from these PBMC. To compare yields, the amount of RNA and DNA was normalized to the number of cells used for extraction. The mean amount of RNA attained from total CPT-PBMC was 0.90 pg/cell, which was similar to 0.86 pg/cell for Ficoll-PBMC (P = 0.871) (Fig. 6a). The average yields of RNA from B cells derived from Ficoll- and CPT-PBMC were 0.73 pg/cell and 0.65 pg/cell, respectively (P = 0.507); CD8+ T lymphocytes, 0.66 pg/cell and 0.50 pg/cell, respectively (P = 0.365); monocytes, 1.43 pg/cell and 1.28 pg/cell, respectively (P = 0.187), and CD4+ T lymphocytes, 0.77 pg/cell and 0.57 pg/cell, respectively (P = 0.266) (Fig. 6a). Similar to RNA yields, there were no significant differences in the yields of DNA (Fig. 7a). Thus, the average amount of DNA extracted from Ficoll-PBMC, and CD19+, CD8+, CD14+ and CD4+ cells derived from these PBMC were 3.76, 5.13, 4.60, 4.01 and 4.42 pg/cell, respectively, compared to 3.74, 5.92, 5.35, 4.03, and 5.71 pg/cell for the same cells derived by CPT isolation protocol (P = 0.977, 0.142, 0.227, 0.955, and 0.141, respectively). Overall, the results indicated that the PBMC isolation method did not affect the number of separated immune cell subsets or the amount of nucleic acids extracted.Fig. 6

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