Gene Expression on the Fluidigm BioMark HD

<strong>Gene</strong> <strong>Expression</strong> on the Fluidigm BioMark HD
Michelle Garred MSc, Paul Lacaze PhD

Overview
• Introduction to Fluidigm (Michelle)
• Advantages of the technology
• Running a Fluidigm gene expression project (Paul)
• Assay design – Taqman vs EvaGreen
• Experimental workflow
• Data analysis
• Case study

BioMark HD Real-Time PCR System
High-throughput qPCR based microfluidic technology for DNA,
RNA, miRNA analysis and next-gen library prep

Advantages of Fluidigm Technology
Network of microfluidic channels,
chambers, and valves
Automatically assemble thousands of
individual PCR reactions
Greatly decrease number of pipetting
steps
Decrease amount of sample and
reagent used

1,300

Technology

Dynamic Arrays: <strong>Gene</strong> <strong>Expression</strong>
96.96 48.48
9,216 data points 2,304 data points

Open Nanoflex Valve
Control Line
Fluid Line

Closed Nanoflex Valve
Control Line
Fluid Line
Fluid Line

<strong>Gene</strong> expression studies: more data is better!
Microarray Validation
Large Patient Cohorts
Transcriptome Sequencing Validation

When is Fluidigm the best time choice for
<strong>Gene</strong> <strong>Expression</strong>?
Many samples (batches of 48 or 96)
Easy workflow and quick time to result
Flexibility of assay selection
Ideal for 50-200 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

Fluidigm <strong>Gene</strong> <strong>Expression</strong> Chemistry Options

Text box
Workflow
96 cDNA Samples
(Diluted STA product)
96 Assays
(primer pairs)
9,216 PCR reactions

C t heat map
96 genes
C t
96
Samples

Fluidigm <strong>Gene</strong> <strong>Expression</strong> Project Workflow
Assay design
- Taqman vs EvaGreen
- Pre-existing vs new primers
Order chips and reagents
Reverse Transcribe and PreAmplify samples
Run Fluidigm chips
Analyse data

<strong>Gene</strong> <strong>Expression</strong> Experimnetal Workflow
Assay design
Reverse Transcription
STA cDNA
Prepare
reagents
and pipet
into chip
Load chip
qPCR on
BioMark
Analyze
data

Chemistry
EvaGreen (SYBRGreen) vs Taqman

Standard EvaGreen ® Chemistry
Denature
Anneal/Amplification
Binds to ANY double stranded DNA target
i.e. NOT sequence specific

Melt Curve Following PCR
Heat 65-95
Product Melts at specific
Tm, Dye falls off

Single Amplicon = Single T m Peak
GAPDH

Evagreen Pros and Cons
• 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

Fluidigm DELTAgene ® Assays
• 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

Taqman Assays

Taqman Assays Pros and Cons
• 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

Specific Target Amplification (STA)
Multiplex
Primer Pool
(0.2X or 500 nM)
+ +
DNA sample
(low conc)
Multiplex PCR
Master Mix
14 cycles
Preamplified
DNA
Dilution
Done in 96 or 384-well plates
“off-chip”

Specific Target Amplification (STA)

BioMark (with STA) has excellent correlation with
the 7900
r= 0.99
ABI 7900
BioMark (with STA)

<strong>Gene</strong> <strong>Expression</strong> Case study
• Data required for NHMRC grant deadline in 3
weeks
• 48 primer pairs purchased (EvaGreen),
• 10-pack of Fluidigm 96.96 GE chips purchased,
with primers – arrived in ~2 weeks
• 48 assays run in duplicate on 96.96 IFC across 96
clinical samples

Case study
48 genes x2
96 Samples

Case study
• Entire project finished in 1 day
~5/48 assay failures (no prior testing)
• Huge saving in reagents, plastics, lab time
• Data ready for grant application deadline

Key Fluidigm Summary Points
• 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

Scientific Applications Talk
Breast Cancer <strong>Gene</strong> <strong>Expression</strong> Profiling
of Many Cells to Single-Cells
Dr Bhupinder Pal
Breast Cancer Laboratory
Walter and Eliza Hall Institute of Medical Research
Melbourne, VIC

Thank you!
Questions?
Paul Lacaze
placaze@mscience.com.au

miRNAs
• RT step is target/miRNA specific
- only miRNA reverse transcribed
• PreAmp using Megaplex primers or targetspecific
primers
• Lab workflow same as <strong>Gene</strong> <strong>Expression</strong>

miRNAs

Reproducibility of <strong>Expression</strong> 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 <strong>Expression</strong> 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 gene
Enough 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)