miRNA Analysis on a Flow Cytometer?

Tom KellyDirector of Sales

Daniel C. Pregibon, Ph.D.Co-Founder and CTO

• 30 Minute presentation

• 15 Minute Question and Answer session

• Telephone lines will be muted until Q&ASession

Webinar Format

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• 30 Minute presentation

• 15 Minute Question and Answer session

• Telephone lines will be muted until Q&ASession

Technology Applications

Presentation Outline

Sample Types

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Terminology

• Multiplexed Assay: An assay that simultaneously measuresmultiple analytes in a single sample.

• miRNA: short (21-23 nucleotides) RNAs that play a major role inregulating gene expression.

• Crude Samples: Minimally processed samples of biologic origin(lysates, homogenates, serum, plasma, etc).

• Open Platform: A technology or application that can be performedon a wide variety of commercially available instruments.

• Encoded Hydrogel Particle: Firefly BioWorks underlying technologythat enables multiplex analyte detection on a wide variety ofinstrumentation.

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• Multiplexed Assay: An assay that simultaneously measuresmultiple analytes in a single sample.

• miRNA: short (21-23 nucleotides) RNAs that play a major role inregulating gene expression.

• Crude Samples: Minimally processed samples of biologic origin(lysates, homogenates, serum, plasma, etc).

• Open Platform: A technology or application that can be performedon a wide variety of commercially available instruments.

• Encoded Hydrogel Particle: Firefly BioWorks underlying technologythat enables multiplex analyte detection on a wide variety ofinstrumentation.

Multiplexed Assays

Less than 10 targetsLow-Plex

ELISA, qPCR, Flow Cytometry, Bead Assays, etc.Technologies

More than 1,000 targetsHigh-Plex

Microarrays, Sequencing, etc.Technologies

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10 to 1,000 targetsMid-Plex

Bead Assays, Digital Hybridization, etc.Technologies

Less than 10 targetsLow-Plex

ELISA, qPCR, Flow Cytometry, Bead Assays, etc.Technologies

Multiplexed Assays

More than 1,000 targetsHigh-Plex

Microarrays, Sequencing, etc.Technologies

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10 to 1,000 targetsMid-Plex

Bead Assays, Digital Hybridization, etc.Technologies

Universal Approach

Technology

Optical Liquid Stamping

Photolithography MicrofluidicsEncoded

Hydrogel Particles

+ =

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The Hydrogel Advantage

VS

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• Hydrophilic -- minimizes non specific interactions• Porous 3D volume providing higher target binding capacity• Thermodynamics for better sensitivity and specificity• Easily incorporates various encoding schemes (graphical or spectral)

VS

Candidate Platforms

High-ThroughputArray Systems

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Microscopesand OtherImagingSystems

FlowCytometers

Candidate Platforms

High-ThroughputArray Systems

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Microscopesand OtherImagingSystems

FlowCytometers

Open Platform

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miRNA Profiling

Application Focus

miRNA Description

• 21-23 nucleotides• Major role in gene

expression and proteintranslation

• Regulate celldevelopment

• Tissue-specificexpression

• Detectable and stable inBlood

• Great diagnosticpotential

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• 21-23 nucleotides• Major role in gene

expression and proteintranslation

• Regulate celldevelopment

• Tissue-specificexpression

• Detectable and stable inBlood

• Great diagnosticpotential

miRNA Profiling

Historical Challenges FirePlex™ miRSelectStudy size can vary greatly Scalable Solution

Complex workflows Minimal processing

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Complex workflows Minimal processingShort, thus difficult to detect Hydrogels are ideally suited for

detecting short nucleic acidsVendor defined miRNA panels User selected targets

Expense Pay only for targets of interest

miRNA DetectionTechnologies

InstrumentIndependent Multiplexed High-

throughputCrude

samplesCost perSample

PCR $

16

Digital Counting $$

Micro-Arrays $$$

Sequencing $$$

miRNA DetectionTechnologies

InstrumentIndependent Multiplexed High-

throughputCrude

samplesCost perSample

PCR $

17

Digital Counting $$

Micro-Arrays $$$

Sequencing $$$

FirePlex™miRSelect $

AdvantageAdvantage FirePlexFirePlex™™

Simple Workflow

45 MinutesRT Shaking

90 Minutes37º Shaking

45 MinutesRT Shaking

Add RunBuffer

AddLabel

AddReporter

RUN3.5 hour protocol (30 min hands on)

RUN3.5 hour protocol (30 min hands on)

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Add RunBuffer

AddLabel

AddReporter

SCAN1 min per well

SCAN1 min per well

ANALYZEFireCode Software

ANALYZEFireCode Software

In the Well

• Up to 25 unique barcodedparticle types

• Each particle type contains theunique complimentary sequencefor the user selected miRNA

• Multiple copies of each particlein every well.

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• Up to 25 unique barcodedparticle types

• Each particle type contains theunique complimentary sequencefor the user selected miRNA

• Multiple copies of each particlein every well.

Molecular Workflow

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Hybridize Label Report

• Probes have miRNA-specific region and universal adapter-specific region

• Two levels of specificity built in:• Stringent hybridization• Terminal sequence alignment during labeling

• Provides discrimination against precursors

Interpreted Data

FirePlex™ on Flow

Raw Data

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Data Collection• Striped particles look like a series of cells to a cytometer.• Simplest machine configuration: Blue laser with green,

yellow, red detectors.• Events are re-grouped by our FireCode software.

FireCode™ Software

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Open Platform

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BDLSR Fortessa

FACSCanto

Accuri C6

MilliporeGuava 8HT

Guava 6HT

MiltenyiMACSQuant

Life TechAttune

TaqMan PCR Correlation

• Example: Detected miRNAdysregulation in several solidtumors.

• Showed great correlation withTaqMan qPCR for samesamples.

• Collaborators have shownconsistency with microarrays,qPCR, and sequencing.

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• Example: Detected miRNAdysregulation in several solidtumors.

• Showed great correlation withTaqMan qPCR for samesamples.

• Collaborators have shownconsistency with microarrays,qPCR, and sequencing.

Firefly ProductsVacuumManifold

FirePlex™

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FireCode ™

Customization Wizardwww.fireflybio.com/search

• Type in any indication, microRNA, etc.

• Wizard searches literature and displaysassociation cloud and miRNA cloud.

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• View annotated list ofreference.

• Build custom panels directlyfrom cloud or furthercustomize.

Total RNA and Crude Samples

Sample Options

Sample Sources

Tissue HomogenateTissue HomogenateCell CultureCell Culture

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FFPE TissueFFPE Tissue

PAXGene BloodPAXGene Blood Serum/ PlasmaSerum/ Plasma

Tissue HomogenateTissue HomogenateCell CultureCell Culture

Sample Guidelines

Sample Type Recommended InputTotal RNA 300 to 1000 ng

Cell Culture 10K to 1M cellsTissue 2 to 10 mg

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Tissue 2 to 10 mgFFPE 2 to 10 mg

Serum 50 to 200 uLPlasma 50 to 200 uL

Cell Lysate

Breast Cancer Cell Lines: Detection in purified RNA vs. lysate.

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PAXgene Blood• 200ng of Total RNA purified from

PAXgene blood.• Total of 80 samples, 12 in

duplicate.• Great reproducibility for

duplicates.

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• 200ng of Total RNA purified fromPAXgene blood.

• Total of 80 samples, 12 induplicate.

• Great reproducibility forduplicates.

Serum/Plasma

• Method co-developed with thelaboratory of Victor Ambros

• 30 minute prep with minimalhands-on time

• microRNA signals significantlyenhanced over other kits

• Compatible with heparinizedblood samples

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• Method co-developed with thelaboratory of Victor Ambros

• 30 minute prep with minimalhands-on time

• microRNA signals significantlyenhanced over other kits

• Compatible with heparinizedblood samples

Platform ComparisonCase Study: 90 Samples for 23 miRNAS

Isolate RNA RNA tocDNA

TargetAmplification Time Cost

TaqMan®PCR(Single Instrument) Required Required Required 10 hours $15K

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Digital Counting NA NA NA 48 hours $25K

MicroArrays Required PlatformDependent

PlatformDependent >48 hours About

$30k

Sequencing(Core Lab) Required Required Required About a

monthMore than

$200K

FirePlex™miRSelect NA NA NA 5 hours $2,250

Technology Applications

Review

Sample Types

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Q&A Session

•15 Minute Session•All lines will remain muted•Please raise hand in GoToWebinar Console

•15 Minute Session•All lines will remain muted•Please raise hand in GoToWebinar Console

By Firefly BioWorks

FirePlex™ miRSelect

Tom KellyDirector of Salestom_kelly@fireflybio.com978-994-9859

Daniel C. Pregibon, Ph.D.Co-Founder and CTOpregibon@fireflybio.com

Protein Detection

Cell Signaling TechnologiesCollaboration:

Performance equivalentto market leader

Attachment chemistry optimized to port existingantibody-based tests onto Firefly platform

FireFly (without serum matrix)

0

1

10

100

1,000

10,000

100,000

1 10 100 1,000 10,000 100,000input antigen (pg/mL)

sign

al InsuliinMCP-1IL-6

Samples run by third party scientist

3-plex on Firefly Platform

Cell Signaling TechnologiesCollaboration:

FireFly (without serum matrix)

0

1

10

100

1,000

10,000

100,000

1 10 100 1,000 10,000 100,000input antigen (pg/mL)

sign

al InsuliinMCP-1IL-6

Luminex (without serum matrix)

1

10

100

1,000

10,000

100,000

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

input antigen (pg/mL)

sign

al InsuliinMCP-1IL-6

3-plex on Luminex Platforminput

input

Detection of phosphorylationwith EGF Activation