Gene Expression on the Fluidigm BioMark HD
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1 Gene Expression on the Fluidigm BioMark HD
2 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
3 BioMark HD Real-Time PCR System High-throughput qpcr based microfluidic technology for DNA, RNA, mirna analysis and next-gen library prep
4 Technology
5 Advantages of Fluidigm Automate thousands of individual PCR reactions Greatly decrease number of pipetting steps Decrease amount of sample and reagent used
6 1,300
7 54
8 Outline
9 Dynamic Arrays: Gene Expression ,216 data points 2,304 data points
10 Open Nanoflex Valve Control Line Fluid Line
11 Closed Nanoflex Valve Control Line Fluid Line Fluid Line
12
13
14 Gene expression applications Microarray Validation Large Patient Cohorts Transcriptome Sequencing Validation
15 When is Fluidigm the best time choice for Gene Expression? Many samples (batches of 24, 48, 96 samples) Easy workflow and quick time to result Flexibility of assay selection Ideal for 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
16 "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."
17 Fluidigm Gene Expression Chemistry Options
18 Text box Workflow 96 cdna Samples (Diluted STA product) 96 Assays (primer pairs) 9,216 PCR reactions
19 C t heat map 96 genes C t 96 Samples
20 Fold-change heat map 96 genes C t 96 Samples
21 Fluidigm Gene Expression 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
22 Gene Expression Experimnetal Workflow Assay design Reverse Transcription STA cdna Prepare reagents and pipet into chip Load chip qpcr on BioMark Analyze data
23 Chemistry EvaGreen (SYBRGreen) vs Taqman
24 Standard EvaGreen Chemistry Denature Anneal/Amplification Binds to ANY double stranded DNA target i.e. NOT sequence specific
25 Melt Curve Following PCR Heat Product Melts at specific Tm, Dye falls off
26 Single Amplicon = Single T m Peak GAPDH
27 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
28 Text boxfluidigm custom assay design service
29 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
30 Taqman Assays
31 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
32 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
33 Specific Target Amplification (STA)
34 BioMark (with STA) has excellent correlation with the 7900 r= 0.99 ABI 7900 BioMark(with STA)
35
36 Workflow: Fast and Easy Prime Pipette Load PCR/Scan 10 min 20 min 55/90 min 90/130 min
37 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
38 mirnas RT step is target/mirna specific - only mirna reverse transcribed PreAmp using Megaplex primers or targetspecific primers Lab workflow same as Gene Expression
39 Case study 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 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
40 16 mirna x6 Cancer Normal Cell line
41 Thank you! Questions? Paul Lacaze
42 mirnas
43 Reproducibility of Expression Levels between Fluidigm and ABI 7900 HT -Tested reproducibility of mirnaexpression by comparing Ct values between ABI 7900 HT and Fluidigm -Mean Ct values between the platforms were Fluidigm (± 0.49) CV=0.08 ABI 7900 HT (±0.82) CV= Significant increase in sensitivity by microfluidics when qpcr reactions are being carried out in nanolitervolumes.
44 Reproducibility of Expression Levels between Fluidigm and ABI 7900 HT
45 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)
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