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Gene Expression on the Fluidigm BioMark HD
Gene Expression on the Fluidigm BioMark HD
• Introduction to Fluidigm James Miller
• Advantages of the technology
• Running a Fluidigm gene expression project Paul Lacaze
• Assay design, chemistry, experimental workflow
• Data analysis
• Case study – miRNA expression in plasma
• Scientific Applications Talk Chris Hanh
Overview
BioMark HD™ Real-Time PCR System
High-throughput qPCR based microfluidic technology for DNA,
RNA, miRNA analysis and next-gen library prep
Technology
Advantages of Fluidigm
Automate thousands of individual
PCR reactions
Greatly decrease number of
pipetting steps
Decrease amount of sample and
reagent used
1,300
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Outline
Dynamic Arrays: Gene Expression
96.96 48.48
9,216 data points 2,304 data points
Open Nanoflex Valve
Fluid Line
Control Line
Closed Nanoflex Valve
Fluid LineFluid Line
Control Line
Gene expression applications
Microarray Validation Large Patient Cohorts
Transcriptome Sequencing Validation
Many samples (batches of 24, 48, 96 samples)
Easy workflow and quick time to result
Flexibility of assay selection
Ideal for 20-200 genes
qPCR vs Microarrays/RNA-seq- qPCR is more sensitive, higher dynamic range
- qPCR is often required for validation
- qPCR gives more rapid result, easier analysis
When is Fluidigm the best time choice for
Gene Expression?
http://www.biomedcentral.com/1471-2164/12/144
"Fluidigm uses the quantitative PCR assays which are
highly sensitive with a dynamic range of at least 6-7
logs[19,22].
The dynamic range of the microarray is usually 3 to 4
logs[25,26]. In our hands, the maximum fold change
observed was around 3 for microarrays and 13 for
Fluidigm dynamic array."
Fluidigm Gene Expression Chemistry Options
Text box
96 cDNA Samples(Diluted STA product)
96 Assays
(primer pairs)
Workflow
9,216 PCR reactions
Ct heat map
96
Samples
96 genes
Ct
Fold-change heat map
96 Samples
Ct
96 genes
Assay design
- Taqman vs EvaGreen
- Pre-existing vs new primers
Order chips and reagents
Reverse Transcribe and PreAmplify samples
Run Fluidigm chips
Analyse data
Fluidigm Gene Expression Project Workflow
Gene Expression Experimnetal Workflow
STA cDNA
Prepare reagents and pipet into chip
Load chipqPCR on BioMark
Analyze data
Assay design
Reverse Transcription
EvaGreen (SYBRGreen) vs Taqman
Chemistry
Standard EvaGreen® Chemistry
Anneal/AmplificationDenature
Binds to ANY double stranded DNA target
i.e. NOT sequence specific
Melt Curve Following PCR
Heat 65-95Product Melts at specific
Tm, Dye falls off
Single Amplicon = Single Tm Peak
GAPDH
• F/R primers with non-specific DNA-binging dye
= More economical than Taqman
• Can be prone to non-specific PCR products
(melt curve required)
• Flexible, easy to change genes in and out
Evagreen Pros and Cons
Text boxFluidigm custom assay design service
• Fluidigm-designed primers for genes/panels
• Minimize upfront cost of assays
• Uses EvaGreen Chemistry
• Amplicons designed to cross an intron
• Assays predicted to multiplex well
Fluidigm DELTAgene® Assays
Taqman Assays
• F/R primers with sequence specific probes
• One probe for each target = specificity
• Less likely to have non-specific PCR products
• More expensive
• Easier to use (all in one tube)
• Many people already have them
Taqman Assays Pros and Cons
Multiplex
Primer Pool(0.2X or 500 nM)
Multiplex PCR
Master Mix DNA sample
(low conc)
14 cycles
Preamplified
DNA
Dilution
+ +
Specific Target Amplification (STA)
Done in 96 or 384-well plates
“off-chip”
Specific Target Amplification (STA)
BioMark (with STA) has excellent correlation with
the 7900
BioMark (with STA)
ABI 7900
r= 0.99
PipettePrime PCR/Scan
20 min10 min 90/130 min
Workflow: Fast and Easy
Load
55/90 min
• Easy conversion from standard qPCR
- Any chemistry can be used
- Miniaturization of reactions
• Samples/assays can be run in replicate to fill the
48 or 96 inlets
• Minimizes pipetting error and lab time
Key Fluidigm Summary Points
• RT step is target/miRNA specific
- only miRNA reverse transcribed
• PreAmp using Megaplex primers or target-
specific primers
• Lab workflow same as Gene Expression
miRNAs
16 Taqman miRNAs
Assays already in lab
Run in n6 = 96 assays
To be detected in varying levels in plasma
Some expressed at very low levels
Candidate early biomarkers of cancer
23 plasma samples
From cancer vs normal patients
Run in n4, plus 4-point std curve = 96 samples
Assays n6 x Samples n4 = n24 replicates
Case study – 96.96 IFCs
16 miRNA x6
Cancer
Cell line
Normal
miRNAs
Reproducibility of Expression Levels between
Fluidigm and ABI 7900 HT
- Tested reproducibility of miRNA expression by comparing Ct
values between ABI 7900 HT and Fluidigm
- Mean Ct values between the platforms were
Fluidigm 12.48 (± 0.49) CV=0.08
ABI 7900 HT 16.21 (± 0.82) CV= 0.06
- “Significant increase in sensitivity by microfluidics when qPCR
reactions are being carried out in nanoliter volumes”.
Reproducibility of Expression Levels between
Fluidigm and ABI 7900 HT
Fluidigm Project planning (Taqman)
Use existing Taqman assays directly on the Fluidigm system
For each Fluidigm IFC inlet, 3ul of Taqman req per geneEnough to measure expression from 48 or 96 samples depending on the chip format (48.48 or 96.96)
If users have less than 48 Taqman assays, that’s fine. Assays can be replicated on the chip as required (12 assays x4, 16 assays x 3 etc).
Empty inlets can also be filled with loading reagent and left blank if required
Users can use any mastermix that is compatible with Taqman chemistry (often the same MM already in their lab)
