Multiplex Assays for Studying Gene Regulation and Cell Function

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

Moderator: Allen Liu MiraiBio Group of Hitachi Software Engineering, Ltd. Presenter: Jim Lazar, PhD OriGene Technologies, Inc.

More than 10,000 customers since 1983
Focus:
MasterPlex – Multiplex analysis software for the Luminex/Bio-Plex xMAP & MSD platforms
ReaderFit – Curve-Fitting ELISA analysis software
DNASIS – Sequence analysis software
Industries: Pharmaceuticals, biotech, academia, government
Geographies: North America, South America, Europe, Asia, Australia
MiraiBio Group of Hitachi Software Engineering, Inc.

Undergraduate degree in Chemistry from Johns Hopkins University
Ph.D. degree in Physical Chemistry from Stanford University
Digene Corporation – Senior Scientist & Director of New Product Development
2002 – Marligen Biosciences
2009 – Marligen acquired by OriGene Technologies – V.P. of Assay Development
Background - James G. Lazar, Ph.D.

Presentation outline:
Intro to Luminex Technology
Multiplexed Genotyping Assays
MicroRNA Expression Profiling
Transcription Factor Profiling
Multiplexed Immunoassays

Missions
Started in1996 with the goal to create largest commercial collection of full-length human cDNAs
Developing technologies focusing on systems biology approaches to study gene function
Developing multiplex assays for every human proteins
About OriGene

25,000 TrueORF cDNA clones
12,000 transfected over-expression lysates
from HEK293 cells
5,000 purified human proteins from HEK293 cells
Develop proteome-wide MRM mass spec standards
Develop mABs for every human full-length proteins
Luminex Multiplex Assays
High density protein/Antigen Chips
Foundations and Technologies

TECHNOLOGY BACKGROUND
What is Luminex xMAP Multiplexing Technology?

Technology platform basics
Industry advances in multiplex and parallel analysis
Test tubes
Microwell plate
Microarray
xMAP ® Technology:
3D liquid bead-array
The Motivation for Multiplexing

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

The Luminex analyzer has two lasers:
Distinguishes the signals from the different colored beads
Measures the signal intensity of the reporter (SAPE) bound
to each bead
Assays Are Read on Luminex™ Analyzer

From Genes to Proteins Genotyping Transcription Factor Profiling microRNA Expression Profiling Immunoassays

Genotyping Multiplex Detection of Single Nucleotide Polymorphisms (SNPs), Insertions and Deletions in Genomic DNA

Genetic variants that can be measured include:
Single Nucleotide Polymorphisms (SNPs)
Small insertions and deletions (indels)
Whole gene deletions
Multiplexed Genotyping Assays

Example in disease predisposition
Screen 9 people for genetic variants involved in diabetes
1 out of 9 shows a genetic profile suggestive for predisposition to diabetes
Closely monitor 1 patient for early signs of disease
Genotyping in Action

PG Panel 1
PG Panel 2
PG Panel 3
PG Panel 4
Obesity Panel 1
Drug Metabolism Panel 1
Alzheimer's Panel 1
Hemochromatosis
Hemoglobinopathies
Cancer Panel 1
Athletic Panel 1
PG Panel 5
PG Panel 6
Alopecia Panel 1
Athletic Panel 2
Y-SNP Genotyping for Ancestry (11 panels)
Beta-Thal (6 panels)
Toll-like Receptors
Current Genotyping Panels

rs2108622 is SNP in the cytochrome P450, CYP4F2 gene
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.
A study of 370 Brazilian ("admixed") patients concludes that prospective CYP4F2 genotyping is of little value to these patients
(presumably due to ethnic-based differences).
rs2108622 – Affecting Optimal Warfarin Dosage

Data for 6 SNPs using MasterPlex™ GT

rs2108622 Population Data (80 samples)

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

MicroRNA Expression Profiling Multiplex Measurement of microRNA Expression Levels

Small 20-24 (avg 22) nucleotide RNAs
Approx 1000 human miRNAs identified
Different sets of expressed miRNAs are found in different cell types and tissues
Regulate gene expression through mRNA silencing
Associated with a wide variety of diseases including cancer, diabetes, heart disease
About microRNA

Recent Headlines

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

MicroRNA Labeling and Detection

OriGene’s focus:
Disease-specific panels
Custom panels:
Create a custom multiplex from any microRNAs in the Sanger Database up to a 100-plex.
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)

No Cross-reactivity Between Unrelated Sequences Cross-hybridization < 1% for all sites

Differential Expression of microRNAs Detected Between Normal and Tumor Samples

Transcription Factor Profiling Multiplex Measurement of Protein-DNA Binding Activity

Transcription factors are proteins that regulate the expression of all genes.
1000-2000 transcription factors encoded in the genome.
Basal TFs comprise the machinery for transcription
Regulatory TFs control gene expression
Both types bind to DNA and form multi-protein complexes
Transcription Factors Control Gene Expression

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

Gel shift assays (EMSAs) and Western blots
gel based, not multiplexed, large sample requirement
Reporter gene assays
requires cell culture, not multiplexed, delayed kinetics
ELISAs
quantitative and automatable, but not easily multiplexed
Traditional Methods for Measuring TF Activity Are Inadequate

Transcription Factor Profiling Assays

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

Slope = 1.07 Lysate Analysis

C U T HL-60 Cells treated with PMA/IM PPAR EMSA C U T

Multiplexed Immunoassays Multiplex Measurement of Protein Concentration in Biological Fluids

Antibody Sandwich Assay Luminex Immunoassays

Produced from TrueORF cDNA clones
5,000 full length human proteins
Expressed in HEK293 cells
Optimal preservation of protein structure, post-translational modifications and functions
Full Length Human Proteins

Dedicated mAb center
Capacity: 200 immunizations per month
Authentic proteins as antigens
Full length proteins made in human cells
Extensive validation
WB: 9 different cell lines
IHC: 24 different tissues
IF
Flow
Luminex
Goal: produce high quality mAb
to every human proteins in 5-10
years
mAb for Every Human Protein

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

Standard Curve from MasterPlex™ QT

Luminex immunoassays
ELISA assay development
ELISA-validated antibody pairs
Antibody coupling to Luminex beads and microplates
Antibody conjugation
Protein coupling to Luminex beads and microplates
ELISA standards
OriGene Immunoassay Portfolio

OriGene offers:
A broad range of Luminex assays
Genotyping Panels
microRNA Detection Panels
Transcription Factor Profiling
Luminex Immunoassays
Extensive tools for custom assay development
Sample testing services
Software partnership with Hitachi
HIGH THROUGHPUT ♦ HIGH PRODUCTIVITY ♦ HIGH VALUE OriGene Immunoassay Portfolio

Topic: Improving your results with MasterPlex QT
Date: July 29 th , 2010
Time: 11:00 AM Pacific / 2:00PM Eastern
Registration:
https://www1.gotomeeting.com/register/351125992
Next Webinar

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shRNA, siRNA, primers
www.origene.com
Summer promotion:
20% off miRNA assays
Jim Lazar: [email_address]

Multiplex Assays for Studying Gene Regulation and Cell Function

by MiraiBio Group of Hitachi Solutions America, Ltd. on Jul 01, 2010

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Multiplex Assays for Studying Gene Regulation and Cell Function — Presentation Transcript