Gene Expression on the Fluidigm BioMark HD · • Introduction to Fluidigm James Miller • Advantages of the technology • Running a Fluidigm gene expression project Paul Lacaze

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

54

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

Thank you!

Questions?

Paul Lacaze

[email protected]

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)