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.

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.

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

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

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

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

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

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

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

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