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
2
• 30 Minute presentation
• 15 Minute Question and Answer session
• Telephone lines will be muted until Q&ASession
Technology Applications
Presentation Outline
Sample Types
3
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.
4
• 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
5
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
6
10 to 1,000 targetsMid-Plex
Bead Assays, Digital Hybridization, etc.Technologies
Universal Approach
Technology
Optical Liquid Stamping
Photolithography MicrofluidicsEncoded
Hydrogel Particles
+ =
8
The Hydrogel Advantage
VS
9
• 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
10
Microscopesand OtherImagingSystems
FlowCytometers
Candidate Platforms
High-ThroughputArray Systems
11
Microscopesand OtherImagingSystems
FlowCytometers
Open Platform
12
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
14
• 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
15
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)
18
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.
19
• 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
20
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
21
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
22
Open Platform
23
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.
24
• Example: Detected miRNAdysregulation in several solidtumors.
• Showed great correlation withTaqMan qPCR for samesamples.
• Collaborators have shownconsistency with microarrays,qPCR, and sequencing.
Firefly ProductsVacuumManifold
FirePlex™
25
FireCode ™
Customization Wizardwww.fireflybio.com/search
• Type in any indication, microRNA, etc.
• Wizard searches literature and displaysassociation cloud and miRNA cloud.
26
• 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
28
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
29
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.
30
PAXgene Blood• 200ng of Total RNA purified from
PAXgene blood.• Total of 80 samples, 12 in
duplicate.• Great reproducibility for
duplicates.
31
• 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
32
• 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
33
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
34
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 [email protected]
Daniel C. Pregibon, Ph.D.Co-Founder and [email protected]
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