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Luminex AssaysHigh-throughput Multiplex Bead Based Assays

Luminex assays are based on xMAP technology (multi-analyte proling beads) enabling

the detection and quantitation o multiple RNA or protein targets simultaneously. The

xMAP system combines a ow cytometer, uorescent-dyed microspheres (beads), lasers

and digital signal processing to efciently allow multiplexing o up to 100 unique assays

within a single sample.

Panomics has an aggressive program or the development o a broad range o assays or

the Luminex platorm and similar instruments based on the xMAP technology. Currently

we have hundreds o RNA targets available in 330 plex assays using our using our

QuantiGene Plex Reagent Systems, with new targets being added weekly. Our Procarta

Human, Mouse and Rat Assays can quantitatively measure cytokines and chemokines

rom a variety o sample sources, including serum and plasma. Weve recently expanded

our amily o assays designed to measure protein expression and monitor protein modi-

cations/activation states in diverse matrices.

We are also proud to be one o the ew Luminex Certied Developers. Luminex have

recognized us as having both unique and extensive assay development capabilities. We

are happy to discuss your specic needs and develop and validate your assay to the same

exacting standards as our commercially available assays.

Luminex Assays romPanomics

Gene Expressionquantitatively

measure up to 30 dierent RNA

transcripts

Transcription FactorProle up to

40 dierent active TFs rom a singlesample

Cytokine/Chemokinesquanti-

tatively measure up to 33 dierent

secreted proteins in serum, plasma

or cell culture supernatant in human

and mouse samples

SH2 DomainsProle phospho-

tyrosine interactions with 30 SH2

protein binding domains

Custom Built AssaysI you cant

nd what youre looking or, we can

custom design and build your assay.

As one o the ew Luminex Certied

Developers, you can be assured o

quality and a speedy response.

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CE

mRNA

LE

BL

CP

Capture

Bead

2.0 Pre-Amplier

2.0 Amplier with

biotinylated Label Probe

Streptavidin Phycoerythrin

a)

b)

Dried Blood Spots

Whole Blood or

PAXgene Blood

FFPE Sections

Animal Tissues

Cultured Cells

Step 1: Release Target RNA

Cells are lysed to release RNA.

Step 3: Signal Amplication

a) Sequential hybridization of the

2.0 Pre-Amplier, 2.0 Amplier

and biotinylated Label Probe,

respectively, for an hour at 50C.

b) Binding with Streptavidin-

conjugated Phycoerythrin (SAPE) at

room temperature for 30 minutes.

Step 4: Detection

The sample is analyzed on a

Luminex* instrument. The level of

SAPE uorescence is proportional

to the amount of mRNA transcripts

captured by the bead.

* Bio-Plex suspension array systemor other Luminex-based array systems.

Step 2: Target RNA Capture

Specic mRNA transcripts are

captured to their respective beads

through a Capture Extender (CE)

Capture Probe (CP) interaction

during an overnight hybridization

at 54C.

QuantiGene Plex 2.0Taking Multiplexed Gene Expression to the Next Level

QuantiGene Plex 2.0 and Luminex

QuantiGene Plex oers high reproducibility and ease-o-use that

make it the perect assay to bridge the technology gap when

studying many genes in a limited number o samples and study-

ing a ew genes in a large number o samples. With QuantiGene

Plex, researchers can easily perorm multiplexed analyses rom rare

or volume-limited samples and can compare results across dier-

ent samples, experiments and laboratories. Proling many genes

simultaneously in a single reaction directly rom cultured cell or

whole blood lysates, or resh, rozen or FFPE tissue homogenates,

can be accomplished without the need or RNA purication,

reverse transcription, or amplication.

QuantiGene Plex 2.0 assays combine branched DNA (bDNA) signal

amplication technology and xMAP (multi-analyte proling) beads

to enable simultaneous quantication o multiple RNA targets

directly rom cultured cell or whole blood lysates; resh, rozen or

ormalin-xed, parafn-embedded (FFPE) tissue homogenates;

or puried RNA preparations. Clinically proven Branched DNA

technology is a sandwich nucleic acid hybridization assay that

provides a unique approach or RNA detection and quantication

by ampliying the reporter signal rather than the target sequence.

By measuring the RNA at the sample source, the assay avoids

variations or errors inherent to extraction and amplication o

target sequences.

Assay Specications

Limit o Detection 2,000 transcripts/assay well

Limit o Quantitation 5,000 transcripts/assay well

Linear Dynamic Range 3 logs

Assay CV 15% intra-assay; 20% inter-assay

Compatible Sample

Types

Cultured cells, whole blood, PAXgene blood

or dried blood spots, resh/rozen tissues,

FFPE samples, puried RNA

Assay Format 96-well plate

Targets/well 330

QuantiGene Plex Applications

Prospective/retrospective studies using whole bloodor FFPE samples

Biomarker validation Predictive toxicology

Microarray validation

Secondary screening

RNAi knockdowns and monitoring of o-target eects

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Demonstrated Performance with Clinically Relevant Sample Types

Using QuantiGene Plex 2.0 in High Throughput ApplicationsMany potential drugs that specically target a particular protein considered to underlie a

given disease have been ound to be less eective than hoped, or to cause signicant side

eects. The intrinsic robustness o living systems against various perturbations is a key actor

that prevents such compounds rom being successul.

By including screening measurements in a more integrated manner using chemical genomic

approaches, i.e. associated pathway elements, dose response, o target targets, the likelihood

o identiying more robust compounds (SMEs) or biologicals that have a higher chance o ulti-

mate success will increase signicantly. At the same time promising candidates that would have

ultimately ailed at a later stage in the development process will be identied during screening,

enabling higher attrition rates supporting the ultimate goal o ailing aster.

QuantiGene Plex 2.0 is ideally suited to deliver cost eective multiplex data or these new

high value contextually relevant assays. Benets to consider:

Target additional pathway elements not just primary genes of interest

Get earlier indication of toxicology proles and stress indicators

Develop dose response proles

384 well or 96 well plate formats are supported

Assay can be readily automated

Our patent pending plex/plex methodology reduces cost and labor considerably

Compatible with the Luminex HTS system

0.1

1

10

100

1000

10000

0.1 1 10 100 1000 10000

QuantiGene Plex

QuantiGenePlex2.0

Housekeeping RNAs

Cytokine RNAs

Values in the graph demonstrate

the expected induction o 4cytokine RNAs in 10 L o whole

blood samples treated with LPS.

No signicant changes were

detected in the expression o 8

housekeeping RNAs. Data rom

QuantiGene Plex 1.0 and

QuantiGene Plex 2.0 assays had a

correlation coefcient o 0.9995.

0

10

20

30

40

50

60

GAPDH

RPLP0

PGK1

HPRT1

PPIB

POLR2A

RPS20

RPL19

RPL32

RPS3

IL-8

Housekeeping RNAsTarget

Fold-change(+PMA/-PM

A)

QuantiGene Plex

QuantiGene Plex 2.0

Values in the graph demonstrate

the expected induction o IL-8

in FFPE preparations o HeLa

cell pellets treated with PMA.

No signicant changes were

detected in the expression o 10

housekeeping RNAs. Data rom

the QuantiGene Plex 1.0 and

QuantiGene Plex 2.0 assays had a

correlation coefcient o 0.9999.

QuantiGene Plex Publications

1. Gupta, A., et al., Role o protein C in renal

dysunction ater polymicrobial sepsis. J

Am Soc Nephrol, 2007. 18(3): p. 860-7.

2. Flagella, M., et al., A multiplex branchedDNA assay or parallel quantitative gene

expression proling. Anal Biochem, 2006.

352(1): p. 50-60.

3. Zheng, Z., Y. Luo, and G.K. McMaster,

Sensitive and quantitative measurement

o gene expression directly rom a small

amount o whole blood. Clin Chem, 2006.

52(7): p. 1294-302.

4. Zhang, A., et al., Small interering RNA and

gene expression analysis using a multi-

plex branched DNA assay without RNA

purication. J Biomol Screen, 2005. 10(6):

p. 549-56.

Assay Highlights

Quantitatively measure multiple

RNA targets simultaneously with

unparalleled accuracy and precision

RNA quantitation directly rom

cultured cells, whole blood, or resh,

rozen or ormalin-xed, parafn-

embedded (FFPE) tissue

No RNA purication No reverse transcription No target amplication

Simple Assay Workfow

Widely used in biomarker validation,

microarray validation, predictivetoxicology and secondary screening

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Procarta Transcription Factor PlexLuminex Assays or Transcription Factor Proling

About TranscriptionFactors

Transcription Factors (TFs) are highly

conserved proteins that bind to DNA and

initiate transcription o a given gene. A

single extracellular stimulus can trigger

multiple signaling pathways, and these in

turn can activate multiple TFs to mediate

the inducible expression o target genes.

The Procarta TF Plex assay is a proling

assay or monitoring the activation o TFs.

We have developed two panels (40-plex

and 43-plex) which are Luminex based

to prole and measure activated TFs.

The 96-well plate ormat enables high-

throughput proling o the DNA binding

activity o TFs in multiple samples with

high sensitivity.

Key Applications

Prole the activities of multiple TFsupon a given drug stimulus

Monitor o-target eects upon agiven drug treatment

Conrm cell signaling pathwaysusing the TF Plex Assay

How It Works

Our novel, Procarta TF assay allows or the proling o multiple TFs rom a variety o sample

types including cell lysates and nuclear extracts. Up to 40 TFs can be analyzed in one well.

bead

1

bead

2

Cis2Cis1

Cis1

Cisn Cis2

Cisn

bead

3

Step 3: Denature cis

elements by heat and

aneal to Luminex beads

with anti-sense sequence

Step 4: Add streptavidin

PE to sample and read on

Luminex machine

Cis1

Cisn

Cisn

Cis2

Cis2

TF2

TFn

TF1

Cis1

Cis1

Cisn

Cis2

TF2

TFn

TF1

Step 1: Incubation of the

cis element probes with the

nuclear extract or whole cell

lysate in 96 well plate

Step 2: Transfer probeTF mixture to separation

plate and wash TF bound

probe mix. Denature cis

elements away from TFs

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TF 40-plex assay: Nuclear Extract from HeLa +/- PMA

MFI

0

2000

4000

6000

8000

10000

12000

14000

Nuclear Extract PMA

Nuclear Extract +PMA

RUNX/AML

AP1

AP2

AR

ATF2

NF-Y

CEBP

FAST1

C-MYB

CREB

E2F1-

1

ELK1

ER

ETS/PEA

FKHR-

1

GATA-

1

GR/PR

HIF-

1

HNF1

IRF1

ISRE

MEF-2

MYOD

NF-1

NFAT

NF-E

1(YY1)

NF-E

2

NFKB1

OCT

P53

PPAR

PAX3

SMAD

STAT1

STAT3

STAT4

NKX-2.5

BRN3

PAX5

Probe only

Procarta TF Plex AssayConrmed by EMSA

The activity o 40 dierent TFs

were proled using the Procarta

Transcription Factor Plex Assay.

Nuclear extracts were prepared

rom serum starved HeLa cells subse-

quently stimulated with PMA or a

vehicle control or 4 hours. Extracts

were used on the Procarta TF Plexassay and the EMSA Gel Shit assays.

1 2 3

AP-1

1 2 3

NF-E2

1 2 3

STAT4

1 2 3

NF-1

Create your own TF Plex Panel

You have the option to either order the ull 40 plex or choose TFs rom either panel to create your own unique 3-39 plex.

RUNX/AML ELK-1 ISRE OCT

AP-1 ER MEF-2 p53AP-2 ETS/PEA MYOD PAX-3

AR FAST-1 NF-1 PAX-5

ATF-2 FKHR-1 NFAT PPAR

BRN-3 GATA-1 NF-E1/YY1 SMAD

CEBP GR/PR NF-E2 STAT-1

C-MYB HIF-1 NFkB STAT-3

CREB HNF1 NKX-2.5 STAT-4

E2F-1 IRF-1 NF-Y STAT-5

Procarta TF Panel 1 (40 TFs)

Visit our website to see the most current list.

Procarta TF Panel 2 (43 TFs)

Create your ownTF Plex Panel

You have the option to

either order the ull plex

sets rom Panel 1 or Panel 2

or choose TFs rom within

either panel to create your

own plex set.

ALF-1/TAL-1 ELF-1 KPF-1 PUR-1

ANTIOXIDENT RE EVI-1 LF-A1 RBAP-1 GAG LVF SIE

AP-4 GFI-1 MRE SRE

CCAAT H4TF MTF SRY

CDP HAS+HBS NEUROD1 TFE-3

CEF-1 HBS/XBP NFkB TR

C-MYC HINF NPAS2 TR(DR-4)

COUP-TF HSF PDX-1 TREF-1/2

E47 IKAROS PIT-1 USF-1

EGR XBP-1 XRE

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Incubate your sample withthe antibody-conjugatedbeads for 30 minutes

IL-10 TNF-

1 Add detection antibody andincubate for 30 minutes

2

Add SAPE and incubatefor 30 minutes

3 Detect interactions ona Luminex instrument

4

Procarta Quantitative, Multiplexed Cytokine/Chemokine Assays

Overview

Procarta Cytokine/Chemokine assays use

the xMAP technology (mutli-analyte

proling beads) to enable the detection

and quantitation o multiple protein

targets simultaneously. The Procarta assay

kits are compatible with all Luminex and

Luminex-based instruments currently

available.

Measure Protein and GeneExpression rom the SameSample Well

Panomics Procarta Cytokine Assay Kits

enable the proling o up to 33 (check

website or an updated list) dierent

cytokines per reaction. When coupled

with Panomics QuantiGene Plex assays,

both gene and protein expression can be

quantitated rom the same sample well

(supernatant or protein quantitation and

cell lysate or mRNA quantitation) enabling

the parallel study o gene expression at

the RNA and protein level.

Procarta Cytokine Assay Kits

Procarta Cytokine assays simultaneously

quantitate cytokines rom diverse matrices

in 3 hours with a sensitivity o 1 pg/mL/

cytokine. Human, Mouse and Rat Cytokine/

Chemokine Assay Kits are available in

1- and 10-plate sizes or xed, o-the-shel

ormats, or By Request, customer dened

mix-to-order ormats. By Request orders

are processed and delivered in a pre-mixedready to use ormat in ~1 week. Procarta

Cytokine Assay Kits contain all the com-

ponents required to process cell culture

supernatant samples and include: pre-

mixed, ready to use, antibody-conjugated

beads; 96-well lter plate and holder;

assay and wash buers; sample buer;

pre-mixed, ready to use detection antibod-

ies; Streptavidin-PE (SAPE) uorescence

detection reagent; and premixed antigenstandards.

Procarta Standard Diluent Kits

Procarta Cytokine Standard Diluent Kits

or plasma and serum, sold separately,

contain a single component, species/

matrix-specic Standard Buer, or prepara-

tion o antigen standards and dilution

o experimental samples (i required).

Panomics Procarta Cytokine Standard

Diluent Kits are designed or use withProcarta Cytokine Assay Kits. Using the

appropriate Standard Buer ensures

optimal recovery and sensitivity o the

cytokines being analyzed in a serum or

plasma matrix.

How It Works

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0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

1 10 100 1,000 10,000 100,000

Analyte (pg/ml)

MedianFluorescen

ceIntensity(MFI)

IL-1alpha IL-1beta

IL-2 IL-3

IL-4 IL-5

IL-6 IL-10

IL-12 (p40) IL-12 (p70)

IL-13 IL-17

GM-CSF MIP-1al ph a

EOTAXIN KC

RANTES TNFalpha

IFNgamma

Standard Curves were generated in cell culture media using Panomics 19-plex Procarta Mouse

Cytokine Assay Kit. Each analyte has a sensitivity o 10 pg/mL or less and an assay range over 3 logs.

Assay Highlights

Mix and match to create your own

plex set rom Human, Mouse and Rat

analytes rom the list below

Complete assay in less than 3 hours

Reagents are supplied at 1X

concentration and ready to use

Quantitative measurements rom

cell culture supernatants, serum or

plasma samples

Minimal sample required, only 25 L

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

0 20 30 60 120 240 480 960 1440 2880

Minutes

mRNAExpression(MF

I)

0

500

1000

1500

2000

2500

3000

3500

4000

ProteinExpression(MFI)

Protein IL-8

mRNA IL-8

0

100

200

300

400

500

600

700

800

0 20 30 60 120 240 480 960 1440 2880

Minutes

mRNAExpres

sion(MFI)

0

50

100

150

200

250

300

350

ProteinExpression(MFI)

Protein IL-1

mRNA IL-1

Specications

Sensitivit y 1 pg/mL/cy tokine

Precision Average Inter-assay CV

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The SH2 Domaina Key to UnderstandingPhosphotyrosine-Dependent SignalTransduction

SH2 domains are one o the many protein domain amilies

that mediate protein-protein interactions in signal trans-duction. Like other domains, SH2 domains are dened by

a conserved region o amino acid residues. The olding

characteristics o this sequence o 100-amino acids allow

these domains to specically recognize and bind to

phosphotyrosine-containing ligands.

There are approximately 120 dierent SH2 domains that

bind to 110 dierent proteins in the human genome. These

protein-protein interactions involving phosphotyrosines,

like those made possible by SH2 domains, are a primary

means o recruiting signaling proteins, and thus play a

major role in signal transduction.

SH2 domains can be ound in enzymes, adaptor proteins,

regulatory subunits o signaling proteins, scaold proteins,

transcription actors and oncogenic proteins. These pro-

teins are integral to the signaling process because they act

as adaptors between receptors and downstream signaling

molecules, transmitting signals within cells and regulating

the kinase activity o specic proteins.

Protein phosphorylation is a major conduit o inormation or

cellular responses, and deects in SH2 domain-dependent

signaling are oten directly or indirectly shown to be involvedin human diseases.

Phosphotyrosine Proling Using SH2 Domains

The Procarta SH2 Domain Plex assay is a 30 plex assay

capable o proling identiying dierences o measuring

SH2 proteins that have bound to phosphorylated tyrosine

residues o proteins.

Cos EGF +/- 5ugCos -, 5ug

Cos EGF 5'

Cos EGF 30

Blank

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

1800.0

2000.0

3BP2(7)

ABL2(12)

BTK(18)

GRAP(19)

CRK(20)

CRKL(21)

DAPP1(25)

FYN(26)

GRB10(27)

GRB14(28)

CSK(29)

VAV3(32)

LCK(34)

LCP2(35)

MATK(36)

NSP1(37)

GrB2(38)

P55G-D1(41)

P85A-D1(42)

P85A-D2(43)

P85B-D1(44)

P85B-D2(45)

PLCG1-D1(46)

PTPN11-D2(47)

PTPN6-D2(51)

SOCS2(52)

STAP2(53)

SYK-D2(54)

TNS(55)

SHC1(56)

P

P

P

P

P

P

Ligand Binding

Receptor Tyrosine Kinase

Receptor phosphorylationand recruitment of SH2Protein to the RTK

The specic SH2 protein binds to the phosphorylated receptor

and initiates the cell signaling cascades

SH2-GRB2

SH2-P13K

SH2-PLCY SH2-

Abl

Ligand

Receptor Tyrosine Kinase

Ligand binds to receptor causing

phosphorylation of the receptor

SH2-GRB2

SH2-P13K

SH2-PLCY SH2-

Abl

SH2-GRB2

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Luminex Technology Overview

Luminexs xMAP technology is built on proven, existing

technologyow cytometry, microspheres, lasers, digital signal

processing and traditional chemistrythat have been combined

in a unique way. Featuring a exible, open-architecture design,

xMAP technology can be congured to perorm a wide variety obioassays quickly, cost-eectively and accurately.

Luminex color-codes tiny beads, called microspheres, into 100

distinct sets. Each bead set can be coated with a reagent specic

to a particular bioassay, allowing the capture and detection o

specic analytes rom a sample. Within the Luminex compact

analyzer, lasers excite the internal dyes that identiy each micro-

sphere particle, and also any reporter dye captured during the

assay. Many readings are made on each bead set, urther validat-

ing the results. In this way, xMAP technology allows multiplexing

o up to 100 unique assays within a single sample, both rapidly

and precisely.

Heres How It Works

The Luminex System is a exible analyzer based on the principles

o ow cytometry that is designed to meet the needs o any

size research laboratory. The system enables you to multiplex

(simultaneously measure) up to 100 analytes in a single microplate

well, using very small sample volumes. At Panomics though, we

oer multiplexed solutions o up to 40 dierent analytes in a single

well. The system delivers ast and cost-eective bioassay results

on many assay ormats that Panomics oers which include: gene

expression, transcription actor proling, cytokine proling andSH2 Domain proling.

The Luminex System is the combination o three core xMAP

technologies. The rst is xMAP microspheres, a amily o 100

uorescently dyed 5.6 micron-sized polystyrene microspheres that

act as both the identier and the solid surace to build the assay.

The second is a ow cytometry-based instrument, the Luminex

analyzer, which integrates key xMAP detection components such

as lasers, optics, advanced uidics and high-speed digital signal

processors. The third component is the assays that are designed

around the microspheres.

xMAP Technology

The xMAP technology uses 5.6 micron polystyrene microspheres

which are internally dyed with red and inrared uorophores. Using

dierent amounts o the two dyes or dierent batches o micro-

spheres, up to 100 dierent microsphere sets can be created. Each

bead is unique with a spectral signature determined by its red/

inrared dye mixture. The bead is lled with a specic known ratio

o the two dyes. As each microsphere carries a unique signature,

the xMAP detection system can identiy to which set it belongs.

Thereore, multiplexing up to 100 tests in a single reaction volume

is possible.

Luminex Reader DesignThe Luminex reader combines two lasers, uidics, and real-time

digital signal processing to distinguish up to 100 dierent sets o

color-coded polystyrene beads, each bearing a dierent assay. The

Luminex reader is an essential tool that perorms the key unctions

o this multiplex technology:

The bead is

impregnated

with the dye

mixture

5.6 Microns

Bead Set 26

Bead Set 21

10UniqueInfraredDyeConcentrations 10U

nique

RedD

yeConc

entra

tions

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Luminex PerformanceHighlights

Reduced cost and labor by

multiplexing

Shortened time-to-results by avor-able reaction kinetics o liquid bead

array approach, with smaller sample

requirements

Liquid reaction kinetics give aster,

more reproducible results than with

solid, planar arrays

Focused, exible multiplexing in

the range o 1 to 100 analytes meets

the needs o a wide variety o appli-

cationsprotein expression proling,

ocused gene expression proling

FluidicsThe reader detects individual beads by ow cytometry. The uidics system

o the reader aligns the beads into single le as they enter a stream o sheath uid and

then enter a ow cell. Once the beads are in single le within the ow cell, each bead is

individually interrogated or bead color (analyte) and assay signal strength (PE uorescence

intensity)

LasersThe reader uses a 532 nm green laser (assay laser) is used to excite the PE dye o

the assay (Streptavidin-PE). The 635 nm solid state laser (red classiy laser) is used to excite

the dyes inside the beads to determine their color or region and is also used or doublet

discrimination by light scatter

DetectorsThe reader has our detectors, one or each o the optical paths shown in the

gure below. Detectors are used to measure the uorescence o the assay, to make bead

determination (1-100) and the last to discriminate between single and aggregate beads.

Bead set 12: IL-6

1835 m = 500 IU

Assay

Detec

tor

532nmGreenLaser

635nmRedLaser

BeadDetector2

BeadDetector1

DDDe

tector

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U.S. Corporate Headquarters

Panomics, Inc.

6519 Dumbarton Circle

Fremont, CA 94555

Toll Free: 877 PANOMICS (1.877.726.6642)

Direct: 1.510.818.2600

Fax: 1.510.818.2610

Email: [email protected]



European Headquarters

Panomics Srl

Via Sardegna 1

20060 Vignate-Milano (Italy)

Tel: +39.02.95.360.250

Fax: +39.02.95.360.992




www.panomics.com

6519 Dumbarton Circle

Fremont, CA 94555

Toll Free: 1.877 PANOMICS (1.877.726.6642)

T: 510.818.2600

F: 510.818.2610

www.panomics.com

For pricing and more inormation visit our website at

www.panomics.com or call us at 1.877.726.6642.

We also oer custom services. Contact us or details.

2007 Panomics, Inc. All rights reserved. Panomics is a trademark o Panomics, Inc. Procarta is a registered trademark o Panomics, Inc. QuantiGene an is a registered trademark exclusively licensed to

P i I MAP d L i i t d t d k L i C ti All th t d k b l t th i ti P t #14940

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