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Flow Cytometry Research Group

Flow Cytometry Research Group Overview of the New FCRG and Proposed Cell Sorting (FACS) Microarray study. Why the formation of FRCG?. Flow is a well established Core labs at most Universities Flow is critical to many downstream molecular and biomedical assays

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Flow Cytometry Research Group

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  1. Flow Cytometry Research Group Overview of the New FCRG and Proposed Cell Sorting (FACS) Microarray study

  2. Why the formation of FRCG? • Flow is a well established Core labs at most Universities • Flow is critical to many downstream molecular and biomedical assays • ABRF is incorporating more cell-based assay RG • Light Microscopy RG • Antibody RG • Several attempts over the years to establish a FCRG • Took the right people….

  3. Current Members • Peter Lopez (Chair) NYU Office Collaborative Science Scott Tighe (Chair) Vermont Cancer Center Alan Bergeron DartLab Dartmouth College Andrew Box Stowers InstituteSridar Chittur SUNY Albany Monica DeLay CCHMC Mike Meyer University of Pittsburgh Mr. Hank Pletcher - University of Pennsylvania Tom Neubert (EB ) NYU

  4. The “tentative” 3 Year Plan for the FCRG • Investigate cellular responses to cell sorting on cell • Molecular and Phenotyic • Jet-in-air AND cuvette-based cell sorters • Year 1 • Pilot study with Jurkat Cells • Jet-in-Air sort with two pressures and nozzle sizes with and without UV, 3hr post sort media incubation, Affymetrix Microarray • Year 2-3 • Investigate other cell types • CD34+ • Dendritic • T-cell • Use different analysis methods other than JUST microarray

  5. 2013 Pilot Study The affects of sorting on gene expression of a suspended cell • Grow new vial of Jurkat cell to early Log phase • FACS sorted using a CytomationMoFlo (Andrew Box-Stower’s) • Matched samples for Pre and Post sort • Added equivalent sheath to presort sample to match the sorted sample • Grow both Pre and post sorted sample in RPMI with serum at 37c 5% CO2 for 2 -4hrs • Pellet and add Trizol

  6. Monica Presentation Here

  7. Review of BD Aria Flow Sorter(s)

  8. Sorting Parameters • Samples are matched with adjusted with correct amount of sheath fluid • Sample will be frozen together at -80 followed by a standard Trizol RNA extraction and QCed • Bioanalyzer • Nanodrop • Gene Expression performed using new Affymetrix GeneArray 2.0ST

  9. Comparisons and ContrastsHigh Pressure High pressure control vs Sorted w/wo UV Low Pressure Low Pressure Control vs Sorted w/wo UV High vs Low Pressure control What is the affect of time of waiting Data AnalysisGenespring 12.5 Quantile normalized using Plier Data Filtered to remove probe signals with >25% CV in each condition 1.5 fold differentially expressed and statistically significant (p<0.05)

  10. Timeline Removed cells from main sample on ice prior to each sort condition. Sorting was performed at room temperature Both conditions – sorted 3 replicates and removed cells from the remaining unsorted material for unsorted control samples 70 PSI 50 uM 20 PSI 100 uM

  11. Microarray Results

  12. 3D PCA plot showing different gene detection

  13. High Pressure (60psi/70um) Sorted (-) UV -3.32 down ncrna:snoRNA -2.34 down Small nucleolar RNA, C/D box 116-1 +3.15 up histone cluster 1 +3.45 up miRNA 520e HP Control +3.06 up (in UV) lincRNA-LRRC38-3 Sorted (+) UV -2.50 down Ankyrin -2.28 down ncrna:snRNA -2.22 down 5S ribosomal 426/175 -2.03 down small nucleolar RNA, C/D box 116-23 -1.80 downsmall nucleolar RNA, C/D box 116-11 -1.75 down small nucleolar RNA, C/D box 41 +1.77 up sialic acid acetylesterase +1.87 up ferritin, heavy polypeptide 1 +3.74 up microRNA 520e

  14. Low Pressure Control vs Low Pressure (-) UV-1.75 fold cut off -4.34 down linc-POMZP3 -2.37 down linc-LRRC8D -2.23 down KIAA1731 -2.14 down 5S ribosomal -2.02 down linc-PRH2-1 -1.92 down coiled-coil domain containing 17 -1.83 down Possible miRNA -1.80 down linc-ANO5-3 -1.80 down snoRNA 1.76 up Mdm2, p53 E3 ubiquitin protein ligase 1.76 up poly(A) polymerase beta 1.76 up linc-GTF2H2-2 1.77 up small nucleolar RNA, C/D box 32B 1.78 up linc-STAT4-2 1.8 up linc-SCAMP1-2 1.81 up transforming growth factor, beta 1 1.81 up taste receptor, type 2, member 19 1.83 up CTGLF6 1.83 up fibroblast growth factor 1 1.88 up neurexophilin 1.9 up immunoglobulin heavy variable 1-18 1.91 up linc-ANKRD50-3 1.92 up proline-rich coiled-coil 2A 1.96 up nuclear receptor co-repressor 2 1.97 up coagulation factor VII 1.98 up midnolin 2.01 up microRNA 4644 2.1 up solute carrier family 25 2.12 up linc-ACTL7A-7 2.19 up snRNA

  15. Low Pressure Control vs Low Pressure (+) UV-1.75 fold cut off -2.14 down Tubulin, beta 4A class IVa -1.83 down c-Maf inducing protein -1.80 down CAP-GLY domain containing linker protein 3 -1.80 down polymerase (RNA) II -1.79 down Kruppel-like factor 16 -1.78 down host cell factor C1 (VP16-accessory protein) 1.75 up linc-IGFL3-3 chr19 1.75 up linc-NR3C2-2 chr4: 1.75 up Rho GDP dissociation inhibitor (GDI) gamma 1.82 up Havana:sense_intronic chromosome RNA 1.88 up linc-MTRNR2L1-2 chr17 1.90 up CTAGE family, member 5 1.97 up MPV17 mitochondrial membrane protein-like-MPV17L 2.19 up ncrna:snoRNA

  16. Simple Venn Diagram • Number of RNAs with 2 fold change-up or down • Common RNAs in overlap HP Control to HP+UV HP Control to HP-UV miRNA 520e

  17. What about MiRNA 520e? • Metastasis-suppressive role via down regulation of IL6/8. • Keklikoglou I et al Oncogene. 2012 Sep 13;31(37) • “Most interestingly, 21 miRNAs located in a cluster on chromosome 19 exhibit similar expression levels. A portion of this large cluster has previously been found to be primate-specific and placenta-associated . Among these miRNAs, miR-518b, miR-518c, miR-519b, miR-519c, miR-520a, miR-520c, miR-520e, miR-520g, and miR-524* are over-expressed in undifferentiated hES cells” • JiaqiangRen et al Journal of Translational Medicine 2009, 7:20 • So what does this mean for sorting cd34+ cells in future studies?

  18. What about while the sample sits on ice during a long sort? • We compared the high pressure control to the low pressure control to represent the time the sample sits on Ice during the Sort for a long periodSame sample except on ice for a while and PBS added • Many genes changes • Many things happen • Needs farther study • Needs more replicates

  19. Down Regulated (2-fold) -10.5 jun proto-oncogene -7.7 heat shock 70kDa protein 1B/A| -5.4 inhibitor of DNA binding 2, dominant negative -5.2 FBJ murine osteosarcoma viral oncogene homolog -5.2 serpin peptidase inhibitor, clade H (heat shock protein 47), -5.0 hairy and enhancer of split 1, (Drosophila) -4.7 microRNA 3143 -3.9 growth arrest and DNA-damage-inducible, beta -3.6 distal-less homeobox 2 -3.5 SMAD family member 7 -3.5 early growth response 1 -3.3 serpin peptidase inhibitor, -3.3 ubiquitin C -3.2 v-maf musculoaponeurotic fibrosarcoma oncogene -3.2 heat shock 70kDa protein 6 (HSP70B') -3.1 metallothionein 1E -3.0 solute carrier family 30 (zinc transporter), member 1 -2.9 BCL2-associated athanogene 3 -2.8 hairy/enhancer-of-split related with YRPW motif 1 -2.6 adrenoceptor beta 2, surface -2.6 serum/glucocorticoid regulated kinase 1 -2.6 docking protein 2, 56kDa -2.5 regulator of G-protein signaling 2, 24kDa -2.5 tubulin, beta 4A class IVa -2.5 jun D proto-oncogene -2.4 CAP-GLY domain containing linker protein 3 -2.4 microRNA 4739 -2.4 polymerase (RNA) II (DNA directed) polypeptide A, -2.4 host cell factor C1 (VP16-accessory protein) -2.4 T cell receptor alpha joining 21 -2.4 ataxin 2-like -2.3 microRNA 449c -2.3 receptor-interacting serine-threonine kinase 4 -2.3 THAP domain containing 9 | SEC31 homolog -2.3 myosin, heavy chain 9, non-muscle -2.3 immediate early response 3 -2.3 hemoglobin, alpha 2 | hemoglobin, alpha 1 -2.3 immediate early response 3 -2.3 secretoglobin, family 1D, member 4 -2.2 H1 histone family, member X -2.2 immediate early response 3 -2.2 v-ski sarcoma viral oncogene homolog (avian) -2.1 microRNA 3142 -2.1 ring finger protein 151 -2.1 activating transcription factor 3 -2.1 Kruppel-like factor 16 -2.1 ATPase, Na+/K+ transporting, alpha 3 polypeptide -2.1 protein kinase D2 -2.1 KH-type splicing regulatory protein | microRNA 3940 -2.0 interferon regulatory factor 2 binding protein-like -2.0 zinc finger protein 777 -2.0 sterile alpha motif domain containing 1 -2.0 PR domain containing 9 -2.0 BCL6 corepressor 18 histone 82 ncRNA c/d box, sno, Linc

  20. Up-Regulated 2.2 ring finger protein 146 2.2 chondroitin sulfate proteoglycan 4 pseudogene 2.2 zinc finger protein 117 2.2 baculoviral IAP repeat-containing protein 1-like 2.2 zinc finger protein 681 2.2 G protein-coupled receptor 52 2.2 importin 5 pseudogene 2.2 oculomedin 2.3 zinc finger and BTB domain containing 26 2.3 ASH1L intronic transcript 1 (non-protein coding) 2.3 protein tyrosine phosphatase, non-receptor type 20C 2.3 taste receptor, type 2, member 20 2.3 taste receptor, type 2, member 30 2.4 taste receptor, type 2, member 14 2.4 EYA3 intronic transcript 1 (non-protein coding) 2.5 ZNF346 intronic transcript 1 (non-protein coding) 2.5 RSF1 intronic transcript 2 (non-protein coding) 2.5 chromosome 15 open reading frame 29 2.7 COX assembly mitochondrial protein 1 homolog (S. cerevisiae) 2.7 chromosome 9 open reading frame 3 2.7 glucuronidase, beta pseudogene 3 2.9 taste receptor, type 2, member 19 4.1 ATG10 intronic transcript 1 (non-protein coding) 2.0 zinc finger protein 83 2.0 zinc finger protein 555 2.0 AFF2 intronic transcript 1 (non-protein coding) 2.0 phosphodiesterase 3B, cGMP-inhibited 2.0 taxilin gamma 2, pseudogene 2.0 zinc finger protein 33B 2.0 FBXO36 intronic transcript 1 (non-protein coding) 2.0 RNA, Ro-associated Y4 pseudogene 23 2.0 aminopeptidasepuromycin sensitive pseudogene 2.0 Rho GTPase activating protein 11B 2.0 speedy homolog E7 2.1 chromosome 22 open reading frame 42 2.1 coiled-coil domain containing 71 2.1 zinc finger protein 627 2.1 CD99 molecule pseudogene 1 2.1 family with sequence similarity 133, member B 2.1 regulator of G-protein signaling 11 2.1 family with sequence similarity 45, member A 2.1 EF-hand domain family, member A2 2.1 phosphatidylinositol 3-kinase-related kinase 2.1 chromosome 1 open reading frame 132 2.1 glucuronidase, beta pseudogene 4 2.1 MORC family CW-type zinc finger 3 2.1 coiled-coil domain containing 144A 2.1 tripartite motif containing 61 23 miRNA, 26 linc, 28 ncRNA-miRNA locus, 31 unknown ncRNA, 45 snoRNA, 6 rRNA, 1 C/D box

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