Gene Expression on the Fluidigm BioMark HD

<strong>Gene</strong> <strong>Expression</strong> on the Fluidigm BioMark HD

Overview
• 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

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

Outline

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

Open Nanoflex Valve
Fluid Line
Control Line

Closed Nanoflex Valve
Fluid Line
Control Line
Fluid Line

<strong>Gene</strong> expression applications
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 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

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

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

96
Samples
C t heat map
96 genes
C t

96 Samples
Fold-change heat map
96 genes
C t

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
Binds to ANY double stranded DNA target
i.e. NOT sequence specific
Anneal/Amplification

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

Text boxFluidigm custom assay design service

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)
Preamplified
DNA
Dilution
14 cycles
Done in 96 or 384-well plates
“off-chip”
Multiplex PCR
Master Mix

Specific Target Amplification (STA)

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

Workflow: Fast and Easy
Prime Pipette Load PCR/Scan
10 min 20 min 55/90 min
90/130 min

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

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>

Case study – 96.96 IFCs
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
23 plasma samples
Candidate early biomarkers of cancer
From cancer vs normal patients
Run in n4, plus 4-point std curve = 96 samples
Assays n6 x Samples n4 = n24 replicates

Cancer
Normal
Cell line
16 miRNA x6

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

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)