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 LiuMiraiBio Group of Hitachi Software Engineering, Ltd.

Presenter: Jim Lazar, PhDOriGene Technologies, Inc.

• More than 10,000 customers since 1983• Focus:

MasterPlex – Multiplex analysis software for the Luminex/Bio-Plex xMAP & MSD platformsReaderFit – Curve-Fitting ELISA analysis softwareDNASIS – 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:

1. Intro to Luminex Technology

2. Multiplexed Genotyping Assays

3. MicroRNA Expression Profiling

4. Transcription Factor Profiling

5. 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

2 Dyes are used to create 100 unique color combinations

100 Color Codes = 100 Simultaneous Tests

Beads are coated with reagents and then mixed to make an array

xMAP® Bead Array

Luminex xMAP® Platform

The Luminex analyzer has two lasers:1. Distinguishes the signals from the different colored beads

2. Measures the signal intensity of the reporter (SAPE) bound

to each bead

Assays Are Read on Luminex™ Analyzer

Genotyping

Transcription Factor

Profiling

microRNA Expression

Profiling

Immunoassays

From Genes to Proteins

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 • Drug Metabolism Panel 2• Drug Metabolism Panel 3 • Drug Metabolism Panel 4 • 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)

    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

Allele Calls from MasterPlex GT

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

 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

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.

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.

Cat. No. Product

11831-050Oncology Detection Panel 1 (47-plex)

11832-050Pancreatic Cancer Detection Panel 1 (12-plex)

11833-050Breast Cancer Detection Panel 1 (8-plex)

11834-050Ovarian Cancer Detection Panel 1 (8-plex)

11835-050Cardiac Detection Panel 1 (10-plex)

11836-050Diabetes Detection Panel 1 (11-plex)

11837-050Hypoxia Detection Panel 1 (10-plex)

11838-050Prostate Cancer Detection Panel 1 (18-plex)

miRNA Detection Panels

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

TATA Pol II

TBP (TATA Binding Protein)

TFIIDEukaryotic PromoterExons

mRNA

TATA Pol II

TBP (TATA Binding Protein)

TFIIDEukaryotic PromoterExons

mRNA

Low Expression Levels

High ExpressionLevels

Transcription Factors Bind to DNA to Regulate Transcription

• 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

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Transcription Factor Profile Obtained with Recombinant NF-kB p50 and NF-1

OCT

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HCT116 Control (MFI)

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Lysate Analysis

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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

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

For serum, plasma, & cell culture supernatant

GF Panel Launch – July 2010

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 THROUGHPUT ♦ ♦ HIGH PRODUCTIVITY HIGH PRODUCTIVITY ♦♦ HIGH VALUEHIGH VALUE

OriGene Immunoassay Portfolio

Topic: Improving your results with MasterPlex QT Date: July 29th, 2010Time: 11:00 AM Pacific / 2:00PM EasternRegistration:

https://www1.gotomeeting.com/register/351125992

Next Webinar

Resources

MiraiBio Group – www.MiraiBio.com

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info@miraibio.com

1-888-615-9600 (Toll-free)

OriGene Technologies, Inc.

• Clones, proteins, mAb,

shRNA, siRNA, primers

www.origene.com

• Summer promotion:

20% off miRNA assays

Jim Lazar: jlizar@origene.com