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Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
Multiplex Assays for Studying Gene Regulation and Cell Function
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Multiplex Assays for Studying Gene Regulation and Cell Function

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Please note: This presentation accompanies a recorded webinar at: …

Please note: This presentation accompanies a recorded webinar at:
https://www1.gotomeeting.com/register/347794241

Biomarkers for studying gene regulation and cell function can be efficiently analyzed by multiplexed methods. Dr. Jim Lazar from OriGene Technologies will provide an overview of four different but related detection technologies that can be used to analyze genetic variants, microRNA expression, transcription factor binding, and protein expression on the Luminex xMAP platform. OriGene’s broad panel of assays and tools for discovery, analysis and validation of multiple classes of important biomarkers will allow researcher to develop more accurate descriptions of biologically complex systems.

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  • 1. From Genes to Proteins: Multiplex Assays for Studying Gene Regulation and Cell Function June 30, 2010 This presentation accompanies the recorded webinar at: https://www1.gotomeeting.com/register/347794241
  • 2. Moderator: Allen Liu MiraiBio Group of Hitachi Software Engineering, Ltd. Presenter: Jim Lazar, PhD OriGene Technologies, Inc.
  • 3. <ul><li>More than 10,000 customers since 1983 </li></ul><ul><li>Focus: </li></ul><ul><ul><li>MasterPlex – Multiplex analysis software for the Luminex/Bio-Plex xMAP & MSD platforms </li></ul></ul><ul><ul><li>ReaderFit – Curve-Fitting ELISA analysis software </li></ul></ul><ul><ul><li>DNASIS – Sequence analysis software </li></ul></ul><ul><li>Industries: Pharmaceuticals, biotech, academia, government </li></ul><ul><li>Geographies: North America, South America, Europe, Asia, Australia </li></ul>MiraiBio Group of Hitachi Software Engineering, Inc.
  • 4. <ul><li>Undergraduate degree in Chemistry from Johns Hopkins University </li></ul><ul><li>Ph.D. degree in Physical Chemistry from Stanford University </li></ul><ul><li>Digene Corporation – Senior Scientist & Director of New Product Development </li></ul><ul><li>2002 – Marligen Biosciences </li></ul><ul><li>2009 – Marligen acquired by OriGene Technologies – V.P. of Assay Development </li></ul>Background - James G. Lazar, Ph.D.
  • 5. <ul><li>Presentation outline: </li></ul><ul><ul><li>Intro to Luminex Technology </li></ul></ul><ul><ul><li>Multiplexed Genotyping Assays </li></ul></ul><ul><ul><li>MicroRNA Expression Profiling </li></ul></ul><ul><ul><li>Transcription Factor Profiling </li></ul></ul><ul><ul><li>Multiplexed Immunoassays </li></ul></ul>
  • 6. <ul><li>Missions </li></ul><ul><ul><li>Started in1996 with the goal to create largest commercial collection of full-length human cDNAs </li></ul></ul><ul><ul><li>Developing technologies focusing on systems biology approaches to study gene function </li></ul></ul><ul><ul><li>Developing multiplex assays for every human proteins </li></ul></ul>About OriGene
  • 7. <ul><ul><li>25,000 TrueORF cDNA clones </li></ul></ul><ul><ul><li>12,000 transfected over-expression lysates </li></ul></ul><ul><ul><li>from HEK293 cells </li></ul></ul><ul><ul><li>5,000 purified human proteins from HEK293 cells </li></ul></ul><ul><ul><li>Develop proteome-wide MRM mass spec standards </li></ul></ul><ul><ul><li>Develop mABs for every human full-length proteins </li></ul></ul><ul><ul><li>Luminex Multiplex Assays </li></ul></ul><ul><ul><li>High density protein/Antigen Chips </li></ul></ul>Foundations and Technologies
  • 8. TECHNOLOGY BACKGROUND <ul><li>What is Luminex xMAP Multiplexing Technology? </li></ul>
  • 9. <ul><li>Technology platform basics </li></ul><ul><ul><li>Industry advances in multiplex and parallel analysis </li></ul></ul><ul><li>Test tubes </li></ul><ul><li>Microwell plate </li></ul><ul><li>Microarray </li></ul><ul><li>xMAP ® Technology: </li></ul><ul><li>3D liquid bead-array </li></ul>The Motivation for Multiplexing
  • 10. 100 Color Codes = 100 Simultaneous Tests Beads are coated with reagents and then mixed to make an array xMAP ® Bead Array Luminex xMAP® Platform 2 Dyes are used to create 100 unique color combinations
  • 11. <ul><li>The Luminex analyzer has two lasers: </li></ul><ul><li>Distinguishes the signals from the different colored beads </li></ul><ul><li>Measures the signal intensity of the reporter (SAPE) bound </li></ul><ul><li>to each bead </li></ul>Assays Are Read on Luminex™ Analyzer
  • 12. From Genes to Proteins Genotyping Transcription Factor Profiling microRNA Expression Profiling Immunoassays
  • 13. Genotyping Multiplex Detection of Single Nucleotide Polymorphisms (SNPs), Insertions and Deletions in Genomic DNA
  • 14. <ul><li>Genetic variants that can be measured include: </li></ul><ul><ul><li>Single Nucleotide Polymorphisms (SNPs) </li></ul></ul><ul><ul><li>Small insertions and deletions (indels) </li></ul></ul><ul><ul><li>Whole gene deletions </li></ul></ul>Multiplexed Genotyping Assays
  • 15. <ul><li>Example in disease predisposition </li></ul><ul><ul><li>Screen 9 people for genetic variants involved in diabetes </li></ul></ul><ul><ul><li>1 out of 9 shows a genetic profile suggestive for predisposition to diabetes </li></ul></ul><ul><ul><li>Closely monitor 1 patient for early signs of disease </li></ul></ul>Genotyping in Action
  • 16.  
  • 17. <ul><li>PG Panel 1 </li></ul><ul><li>PG Panel 2 </li></ul><ul><li>PG Panel 3 </li></ul><ul><li>PG Panel 4 </li></ul><ul><li>Obesity Panel 1 </li></ul><ul><li>Drug Metabolism Panel 1 </li></ul><ul><li>Drug Metabolism Panel 2 </li></ul><ul><li>Drug Metabolism Panel 3 </li></ul><ul><li>Drug Metabolism Panel 4 </li></ul><ul><li>Alzheimer's Panel 1 </li></ul><ul><li>Hemochromatosis </li></ul><ul><li>Hemoglobinopathies </li></ul><ul><li>Cancer Panel 1 </li></ul><ul><li>Athletic Panel 1 </li></ul><ul><li>PG Panel 5 </li></ul><ul><li>PG Panel 6 </li></ul><ul><li>Alopecia Panel 1 </li></ul><ul><li>Athletic Panel 2 </li></ul><ul><li>Y-SNP Genotyping for Ancestry (11 panels) </li></ul><ul><li>Beta-Thal (6 panels) </li></ul><ul><li>Toll-like Receptors </li></ul>Current Genotyping Panels
  • 18. <ul><li>rs2108622 is SNP in the cytochrome P450, CYP4F2 gene </li></ul><ul><li>A study of Italian patients concluded that rs2108622 (T:T) patients require 5.49 mg/day of warfarin versus 2.93 mg/day for (C:C) patients. </li></ul><ul><li>A study of 370 Brazilian (&quot;admixed&quot;) patients concludes that prospective CYP4F2 genotyping is of little value to these patients </li></ul><ul><ul><li>(presumably due to ethnic-based differences). </li></ul></ul>rs2108622 – Affecting Optimal Warfarin Dosage
  • 19. Data for 6 SNPs using MasterPlex™ GT
  • 20. rs2108622 Population Data (80 samples)
  • 21. Allele Calls from MasterPlex GT     rs1057910 rs1799853 rs2108622-RS rs4917639-RS rs8050894 rs9923231-RS [C2] 81   A C C A C C [D2] 82   A,C T,C T C G T [E2] 83   A C C A G,C C,T [F2] 84   A C C,T A G,C C,T [G2] 85   A C C,T A G,C C,T [H2] 86   A T,C C,T C,A C C [A3] 87   A C C A G,C C,T [B3] 88   A,C C C,T C,A C C [C3] 89   A C C A G,C C,T [D3] 90   A C C,T A G,C C,T [E3] 91   A C C A G,C C,T [F3] 92   A C C,T A G T [G3] 93   A,C C C,T C,A G T [H3] 94   A C C A G T [A4] 95   A C C,T C,A G,C C,T
  • 22. MicroRNA Expression Profiling Multiplex Measurement of microRNA Expression Levels
  • 23. <ul><li>Small 20-24 (avg 22) nucleotide RNAs </li></ul><ul><li>Approx 1000 human miRNAs identified </li></ul><ul><li>Different sets of expressed miRNAs are found in different cell types and tissues </li></ul><ul><li>Regulate gene expression through mRNA silencing </li></ul><ul><li>Associated with a wide variety of diseases including cancer, diabetes, heart disease </li></ul>About microRNA
  • 24. Recent Headlines
  • 25. Methods for Detecting miRNA Expression # Dependent on labeling method used. Some planar array labeling and detection methods are compatible with total RNA or RNA isolated from FFPE samples. * Time based on microRNA Labeling and Detection Panel protocol.   OriGene microRNA Detection Panels Planar Microarrays qRT-PCR Sample Type       Total RNA YES NO# YES Enriched LMW RNA YES YES YES Degraded RNA (RIN<7) YES NO# YES RT Step NO NO YES Amplification NO NO YES Multiplex YES YES NO <4 hour protocol YES* NO YES Sample Throughput HIGH LOW MEDIUM Flexible assay format YES NO YES
  • 26. MicroRNA Labeling and Detection
  • 27. <ul><li>OriGene’s focus: </li></ul><ul><li>Disease-specific panels </li></ul><ul><li>Custom panels: </li></ul><ul><ul><ul><li>Create a custom multiplex from any microRNAs in the Sanger Database up to a 100-plex. </li></ul></ul></ul>miRNA Detection Panels Cat. No. Product 11831-050 Oncology Detection Panel 1 (47-plex) 11832-050 Pancreatic Cancer Detection Panel 1 (12-plex) 11833-050 Breast Cancer Detection Panel 1 (8-plex) 11834-050 Ovarian Cancer Detection Panel 1 (8-plex) 11835-050 Cardiac Detection Panel 1 (10-plex) 11836-050 Diabetes Detection Panel 1 (11-plex) 11837-050 Hypoxia Detection Panel 1 (10-plex) 11838-050 Prostate Cancer Detection Panel 1 (18-plex)
  • 28. No Cross-reactivity Between Unrelated Sequences Cross-hybridization < 1% for all sites
  • 29. Differential Expression of microRNAs Detected Between Normal and Tumor Samples
  • 30. Transcription Factor Profiling Multiplex Measurement of Protein-DNA Binding Activity
  • 31. <ul><li>Transcription factors are proteins that regulate the expression of all genes. </li></ul><ul><li>1000-2000 transcription factors encoded in the genome. </li></ul><ul><li>Basal TFs comprise the machinery for transcription </li></ul><ul><li>Regulatory TFs control gene expression </li></ul><ul><li>Both types bind to DNA and form multi-protein complexes </li></ul>Transcription Factors Control Gene Expression
  • 32. Low Expression Levels High Expression Levels Transcription Factors Bind to DNA to Regulate Transcription TATA Pol II TBP (TATA Binding Protein) TFIID Eukaryotic Promoter Exons mRNA TATA Pol II TBP (TATA Binding Protein) TFIID Eukaryotic Promoter Exons mRNA
  • 33. <ul><li>Gel shift assays (EMSAs) and Western blots </li></ul><ul><ul><li>gel based, not multiplexed, large sample requirement </li></ul></ul><ul><li>Reporter gene assays </li></ul><ul><ul><li>requires cell culture, not multiplexed, delayed kinetics </li></ul></ul><ul><li>ELISAs </li></ul><ul><ul><li>quantitative and automatable, but not easily multiplexed </li></ul></ul>Traditional Methods for Measuring TF Activity Are Inadequate
  • 34. Transcription Factor Profiling Assays
  • 35. Transcription Factor Profile Obtained with Recombinant NF-kB p50 and NF-1 0 200 400 600 800 1000 1200 NF-kb CREB ISRE EGR ETS alt AP4 NF-1 Octamer GATA alt PPAR AP1 SMAD 1 TATA SMAD 3 STAT NBS1 MTB1b HNF-4 AML-1 FREAK SAF CRE ATF1 TAL1 E2F1-5 E2F6 MYCX NFY1 ER Assay Net MFI StRE
  • 36. Slope = 1.07 Lysate Analysis
  • 37. C U T HL-60 Cells treated with PMA/IM PPAR EMSA C U T
  • 38. Multiplexed Immunoassays Multiplex Measurement of Protein Concentration in Biological Fluids
  • 39. Antibody Sandwich Assay Luminex Immunoassays
  • 40. <ul><li>Produced from TrueORF cDNA clones </li></ul><ul><li>5,000 full length human proteins </li></ul><ul><li>Expressed in HEK293 cells </li></ul><ul><li>Optimal preservation of protein structure, post-translational modifications and functions </li></ul>Full Length Human Proteins
  • 41. <ul><li>Dedicated mAb center </li></ul><ul><ul><li>Capacity: 200 immunizations per month </li></ul></ul><ul><li>Authentic proteins as antigens </li></ul><ul><ul><li>Full length proteins made in human cells </li></ul></ul><ul><li>Extensive validation </li></ul><ul><ul><li>WB: 9 different cell lines </li></ul></ul><ul><ul><li>IHC: 24 different tissues </li></ul></ul><ul><ul><li>IF </li></ul></ul><ul><ul><li>Flow </li></ul></ul><ul><ul><li>Luminex </li></ul></ul><ul><li>Goal: produce high quality mAb </li></ul><ul><li>to every human proteins in 5-10 </li></ul><ul><li>years </li></ul>mAb for Every Human Protein
  • 42. For serum, plasma, & cell culture supernatant GF Panel Launch – July 2010 Analyte Name Symbol Platelet-derived growth factor AB PDGFAB Platelet-derived growth factor BB PDGFBB Fibroblast growth factor 4 FGF4 Vascular endothelial growth factor D VEGFD Fibroblast growth factor 2 FGF2 Epidermal growth factor EGF Hepatocyte growth factor HGF Flt-3 ligand FLT3LG Angiopoietin 2 ANGPT2 Placental growth factor PGF Vascular endothelial growth factor A VEGFA
  • 43. Standard Curve from MasterPlex™ QT
  • 44. <ul><li>Luminex immunoassays </li></ul><ul><li>ELISA assay development </li></ul><ul><li>ELISA-validated antibody pairs </li></ul><ul><li>Antibody coupling to Luminex beads and microplates </li></ul><ul><li>Antibody conjugation </li></ul><ul><li>Protein coupling to Luminex beads and microplates </li></ul><ul><li>ELISA standards </li></ul>OriGene Immunoassay Portfolio
  • 45. <ul><li>OriGene offers: </li></ul><ul><ul><li>A broad range of Luminex assays </li></ul></ul><ul><ul><ul><li>Genotyping Panels </li></ul></ul></ul><ul><ul><ul><li>microRNA Detection Panels </li></ul></ul></ul><ul><ul><ul><li>Transcription Factor Profiling </li></ul></ul></ul><ul><ul><ul><li>Luminex Immunoassays </li></ul></ul></ul><ul><ul><li>Extensive tools for custom assay development </li></ul></ul><ul><ul><li>Sample testing services </li></ul></ul><ul><ul><li>Software partnership with Hitachi </li></ul></ul>HIGH THROUGHPUT ♦ HIGH PRODUCTIVITY ♦ HIGH VALUE OriGene Immunoassay Portfolio
  • 46. <ul><li>Topic: Improving your results with MasterPlex QT </li></ul><ul><li>Date: July 29 th , 2010 </li></ul><ul><li>Time: 11:00 AM Pacific / 2:00PM Eastern </li></ul><ul><li>Registration: </li></ul><ul><li>https://www1.gotomeeting.com/register/351125992 </li></ul>Next Webinar
  • 47. <ul><li>MiraiBio Group – www.MiraiBio.com </li></ul><ul><ul><li>Free Software Trials </li></ul></ul><ul><ul><li>Blog </li></ul></ul><ul><ul><li>Webinars </li></ul></ul><ul><ul><li>Community </li></ul></ul><ul><ul><li>Online Tutorials </li></ul></ul><ul><ul><li>Knowledge Base </li></ul></ul><ul><li>info@miraibio.com </li></ul><ul><li>1-888-615-9600 (Toll-free) </li></ul>Resources <ul><li>OriGene Technologies, Inc. </li></ul><ul><ul><li>Clones, proteins, mAb, </li></ul></ul><ul><ul><li>shRNA, siRNA, primers </li></ul></ul><ul><ul><li>www.origene.com </li></ul></ul><ul><ul><li>Summer promotion: </li></ul></ul><ul><ul><li>20% off miRNA assays </li></ul></ul><ul><li>Jim Lazar: [email_address] </li></ul>

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