The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
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1 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes The effects of Cell Subset Isolation Method on Gene Expression inleukocytes Nadejda Beliakova-Bethell, Marta Massanella et al
2 Isolation of specific subsets of blood cells Positive immunomagentic selection Negative immunomegnetic selection FACS (fluorescence activated cell sorting) Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
3 Hypotheses Positive selection and FACS yield higher purity Positive selection and FACS have an impact on gene expression FACS may upregulate stress response genes Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
4 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes Gene expression signatures to investigate disease pathogenesis effects of therapeutic intervention
5 PBMC gene expression profiling Pro: Ease of sampling Problem: mixture of different cells HIV Systemic lupus erythematosus Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
6 Subsets of blood cells isolated from PBMCs CD4+ -T-cells CD8+-T-cells B-cells Monocytes Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
7 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
8 POSITIVE SELECTION NEGATIVE SELECTION FACS CD4+ T cells Donors 1,2,3,4,5 Donors 1,2,3,4,5 Donors 1,2,3,4,5 CD8+ T cells Donors 1,2,3,4,5 Donors _,2,3,4,5 Donors 1,2,3,4,5 B cells Donors _,2,3,4,5 Donors _,2,3,4,5 Donors 1,2,3,_,_ Monocytes Donors 1,2,3,4,5 Donors 1,_,3,4,5 Donor _,2,_,_,_ Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
9 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes RNA extraction 5 donors, 3 isolation methods, 4 cell types RNA quality measurement RNA integrity number (RIN) Unsupervised clustering
10 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
11 Samples clustered primarily by cell type Secondarily by isolation method Not by donor Significant difference between Isolation method Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
12 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
13 CD4+-cells contaminated with monocytes B-cell samples largest spread between isolation methods CD8+-cells and monocytes tight clusters indicate less effect of contamination and isolation method Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
14 Effect of Isolation method on gene Expression Positive Selection utilizes cell receptor antibodies Potential activation cascade Receptor blockade Negative Selection using antibody coktails FACS antibodies stain detected cells Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
15 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
16 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes Stress genes have the lowest expression in FACS-isolated cells In monocytes isolated by positive and negative selection, platelet contamination affected expression of stress genes
17 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
18 Discussion FACS is the optimal method for PBMC subset separation for gene expression profiling studies High purity Little impact on cellular transcription Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes
19 Nadejda Beliakova-Bethell, Marta Massanella et al The effects of Cell Subset Isolation Method on Gene Expression inleukocytes Exception: Monocytes Low yields were obtained by FACS Concentration of 10millions/ml used Long term effects not examined
20 Thank you for your attention!
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