INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune cell subsets by immunomagnetic separation and the RNA integrity after isolated cells from samples of HIV-infected patients. METHODS: PBMC from Healthy Controls (HC, n=15), Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=15) and HIV-infected patients on therapy (ART, n=15) were isolated by Ficoll-Paque density gradient centrifugation. Subsets were separated with monoclonal antibodies (CD56, CD14, CD4, and CD8) conjugated to microbeads. We evaluated the yield and purity of each subset isolated from PBMCs under resting and activated conditions; LPS, anti-CD3/CD28 and anti-CD16 were used to activate monocytes, PBMC, T cells and NK cells, respectively. The quality of extracted RNA was tested by 2100 Bioanalyzer. RESULTS: In resting conditions, the average yield of CD14+ (monocytes) was decreased (p=0.021) in HIV+ patients compared with healthy controls. CD56+ (Natural Killer-NKs; p=0.03) and CD8+ (Cytotoxic T lymphocytes-CTL p=0.001) cells were increased in HIV+ patients after 72h of activation. The purity assay detected significant differences in CD14+ (p≤0.001) and CD8+ (p=0.034) subpopulations when comparing PBMC isolated either from healthy controls or HIV+ patients. The number of activated cells in HIV+ presented differences in CD8 subset (p=0.003). Finally, similar quantities of high quality RNA were extracted from immune cells subsets obtained by our method. Specifically, we show that Bioanalyzer electrophenograms reveal optimal RIN values in HIV positive and negative patients in resting condition (EC:8;HC:6.5;VC:8.80;VP:8;HAART:7.5) and activated condition (EC:9;HC:6.7;VC:8.2;VP:7.2;HAART:8.6). CONCLUSION: This method allowed us to obtain a sufficient quantity of different isolated immune cell subsets from HIV-infected individuals at different disease stages. Moreover, the assessed qualities of nucleic acids allow us to perform subsequent molecular studies, such as microRNA profiling.
INTRODUCTION: Peripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune cell subsets by immunomagnetic separation and the RNA integrity after isolated cells from samples of HIV-infectedpatients. METHODS: PBMC from Healthy Controls (HC, n=15), Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=15) and HIV-infectedpatients on therapy (ART, n=15) were isolated by Ficoll-Paque density gradient centrifugation. Subsets were separated with monoclonal antibodies (CD56, CD14, CD4, and CD8) conjugated to microbeads. We evaluated the yield and purity of each subset isolated from PBMCs under resting and activated conditions; LPS, anti-CD3/CD28 and anti-CD16 were used to activate monocytes, PBMC, T cells and NK cells, respectively. The quality of extracted RNA was tested by 2100 Bioanalyzer. RESULTS: In resting conditions, the average yield of CD14+ (monocytes) was decreased (p=0.021) in HIV+ patients compared with healthy controls. CD56+ (Natural Killer-NKs; p=0.03) and CD8+ (Cytotoxic T lymphocytes-CTL p=0.001) cells were increased in HIV+ patients after 72h of activation. The purity assay detected significant differences in CD14+ (p≤0.001) and CD8+ (p=0.034) subpopulations when comparing PBMC isolated either from healthy controls or HIV+ patients. The number of activated cells in HIV+ presented differences in CD8 subset (p=0.003). Finally, similar quantities of high quality RNA were extracted from immune cells subsets obtained by our method. Specifically, we show that Bioanalyzer electrophenograms reveal optimal RIN values in HIV positive and negative patients in resting condition (EC:8;HC:6.5;VC:8.80;VP:8;HAART:7.5) and activated condition (EC:9;HC:6.7;VC:8.2;VP:7.2;HAART:8.6). CONCLUSION: This method allowed us to obtain a sufficient quantity of different isolated immune cell subsets from HIV-infected individuals at different disease stages. Moreover, the assessed qualities of nucleic acids allow us to perform subsequent molecular studies, such as microRNA profiling.
Authors: Julia Alles; Nicole Ludwig; Stefanie Rheinheimer; Petra Leidinger; Friedrich A Grässer; Andreas Keller; Eckart Meese Journal: Oncotarget Date: 2017-05-07
Authors: K M Santos; I N F Gomes; R J Silva-Oliveira; F E Pinto; B G Oliveira; R C R Chagas; W Romão; R M V Reis; R I M A Ribeiro Journal: Biomed Res Int Date: 2018-03-25 Impact factor: 3.411