Multiplex Assays for Studying Gene Regulation and Cell Function
-
Upload
miraibio-group-of-hitachi-solutions-america-ltd -
Category
Technology
-
view
1.356 -
download
0
description
Transcript of 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
0
200
400
600
800
1000
1200N
F-k
b
CR
EB
ISR
E
EG
R
ET
S a
lt
AP
4
NF
-1
Oct
amer
GA
TA
alt
PP
AR
AP
1
SM
AD
1
TA
TA
SM
AD
3
ST
AT
NB
S1
MT
B1b
HN
F-4
AM
L-1
FR
EA
K
SA
F
CR
E A
TF
1
TA
L1
E2F
1-5
E2F
6
MY
CX
NF
Y1
ER
Assay
Net
MF
I
StR
E
Transcription Factor Profile Obtained with Recombinant NF-kB p50 and NF-1
OCT
0
2000
4000
6000
8000
10000
12000
14000
0 1000 2000 3000 4000 5000 6000 7000 8000
HCT116 Control (MFI)
Oct
-Tra
nsf
ecte
d (
MF
I)
Slope = 1.07
Lysate Analysis
C U T
Sterol SRE
GATA
OCT
CRE-ATF
PPAR
MEF2
CREB
MYC-MAX
NF1
0
500
1000
1500
2000
0 50 100 150 200 250 300 350 400 450 500
Untreated HL60 (MFI)
Tre
ate
d H
L6
0 P
MA
(M
FI)
PPAR EMSAC U T
HL-60 Cells treated with PMA/IM
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
• Free Software Trials
• Blog
• Webinars
• Community
• Online Tutorials
• Knowledge Base
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: [email protected]