Moving from qpcr Assays to qpcr Arrays. Ken Livak qpcr 2009, Freising-Weihenstephan 11 March 2009

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1 Moving from qpcr Assays to qpcr Arrays Ken Livak qpcr 2009, Freising-Weihenstephan 11 March 2009

2 qpcr Technical Milestones Nuclease Assay Holland et al., Proc Natl. Acad. Sci. 88: Real Time PCR Higuchi et al., Bio/Technology 10: Fluorogenic Probe Lee et al., Nucleic Acids Res. 21: Quencher Livak et al., PCR Meth. Appl. 4:357 2

3 TaqMan Technology Introduced May, 1996 R Q 5' 3' 3' 5' 5' 3' 5' ABI PRISM

4 Advantages of TaqMan Quantification Sensitivity Specificity Detection requires hybridization with 3 independent oligos Large dynamic range Arrays limited to 2-3 orders of magnitude Greater reproducibility Microarrays are discovery tools TaqMan assays are analysis tools 4

5 Paradigm for Gene Expression Analysis Use microarrays on a few samples to survey a large number of genes in order to identify candidates that may have significant variation Use qpcr on a much larger number of samples to analyze the candidate genes in order to validate which ones are truly significant 5

6 # genes per assay Gene Expression 100,000 Hybridization Arrays Next Gen Sequencing 10,000 1,000 qpcr Arrays , ,000 # samples qpcr Assays 6

7 Fluidigm s Objective: Revolutionize Life Sciences with the IC for Biology For Decades: The Tyranny of Pipetting impedes a $35Bn Industry Fluidigm s IFCs deliver the promise of microfluidics: Integrated Circuits for Biology 7

8 Fluidigm Commercialized the Fluidic Equivalent of a Transistor Transistor: Fluidigm s Nano-Flex valve: Enables integrated electronic circuit Enables integrated fluidic circuit 8

9 Nano-Flex Valve: Open 9

10 Nano-Flex Valve: Closed 10

11 Fluidigm s IFC Technology Delivers Unrivaled Integration for Fluidics 3,000 Valves / cm

12 The BioMark System for High- Throughput Gene Expression 12

13 96.96 Dynamic Array Nanofluidic chip Samples Assays 13

14 96.96 Dynamic Array 96 Samples 96 Assays 96 user dispensed primer-probe assays Each chip is user configurable at the time of the experiment, no prespotting is required Throughput 9,216 PCR reactions per dynamic array Standard SBS format for fast and easy dispensing 14

15 Why the Dynamic Array 200X Less!!! 384-well Dynamic Array Master Mix 46mL 240µl Primer-probe 9.2mL 480µl Plates 24 1 Time 8 days 3 hours Pipette steps 18, Example: 96 samples vs 96 genes on a Dynamic Array 15

16 How Dynamic Arrays Stack Up Time Labor Reagents 16

17 Gene Expression Discovery for Cancer Diagnostics: Myriad Genetics ~1000 genes across thousands of samples Gold-standard performance at microarray density A single platform for discovery, validation and application On precious samples Paraffin embedded tissue samples Needle biopsies 17

18 Barriers to Single-Cell Gene Expression Analysis Microarrays Insufficient Sensitivity Only more abundant transcripts detected, whereas fluctuations in rarer transcripts are more likely to generate significant differences among cells Cumbersome Workflow Precludes facile analysis of multiple samples qpcr Too Expensive Cost of assay-specific and PCR reagents for multiple samples by multiple assays is prohibitive Arduous Pipetting Necessitates robots and lots of plastic (reaction plates & pipet tips) 18

19 Paradigm for Gene Expression Analysis Microarrays for discovery qpcr for detailed analysis How can the survey step be done with single cells or other limited samples? 19

20 20 Matrixed Primer Concept R1,R5,R9,R13 P1 R2,R6,R10,R14 P2 R3,R7,R11,R15 P3 R4,R8,R12,R16 P4 F1,F2,F3,F4 F1,F2,F3,F4 R1,R5,R9,R13 P1 F1,F2,F3,F4 R2,R6,R10,R14 P2 F1,F2,F3,F4 R3,R7,R11,R15 P3 F1,F2,F3,F4 R4,R8,R12,R16 P4 F5,F6,F7,F8 F5,F6,F7,F8 R1,R5,R9,R13 P1 F5,F6,F7,F8 R2,R6,R10,R14 P2 F5,F6,F7,F8 R3,R7,R11,R15 P3 F5,F6,F7,F8 R4,R8,R12,R16 P4 F9,F10,F11,F12 F9,F10,F11,F12 R1,R5,R9,R13 P1 F9,F10,F11,F12 R2,R6,R10,R14 P2 F9,F10,F11,F12 R3,R7,R11,R15 P3 F9,F10,F11,F12 R4,R8,R12,R16 P4 F13,F14,F15,F16 F13,F14,F15,F16 R1,R5,R9,R13 P1 F13,F14,F15,F16 R2,R6,R10,R14 P2 F13,F14,F15,F16 R3,R7,R11,R15 P3 F13,F14,F15,F16 R4,R8,R12,R16 P4

21 Probes for Matrixed Primer Assays Roche Universal ProbeLibrary 165 hydrolysis probes selected to detect 8- and 9-mer motifs that are common in the human and other transcriptomes Enabled by use of LNA analogs to maintain high T m Within human transcriptome, each probe binds to approx transcripts, while each transcript is detected by approx. 16 different probes Labeled at the 5' end with fluorescein (FAM) and at the 3' end with a dark quencher dye 21

22 Samples Assays

23 1152 Human Assays in a Array 1152 assays were designed for 997 genes selected from the MAQC study These assays were configured into an array with 24 rows and 48 columns 23

24 Matrixed Primer Workflow Design Pool genes by common UPL probe (48 pools) Design primers to amplify segment containing required probe Construct Assay Pools Order primers (2304 oligos) Pool primers and probes in appropriate configurations 24 Pools, each pool containing 48 Forward Primers 48 Pools, each pool containing 24 Reverse Primers + 1 Probe For Specific Target Amplification, 1 Pool containing all 2304 Primers 24

25 Matrixed Primer Workflow Run Experiment Specific Target Amplification (1152-plex) of cdna samples Brain & UHR Partition each sample to mix with the 24 pools of Forward Primers plus PCR Master Mix. Dispense into the Sample Inlets of a Dynamic Array. Dispense 48 pools of Reverse Primers plus Probe into the Assay Inlets Load the Dynamic Array and perform realtime PCR 25

26 Why Does Preamplification Work Like multiplex PCR in general, primer mischief products are generated during preamplification Low primer concentrations (50 nm) reduce, but do not eliminate, nonspecific amplification The limited number of cycles used in preamplification attenuates any competition that might occur between specific and nonspecific products Thus, specific products are amplified at near 100% efficiency despite the generation of primer mischief products In the final detection reaction, only a single primer pair is present, so the chance of amplifying a primer mischief product from the preamplification is slight Although this is counterintuitive, the chance of amplifying a primer mischief product decreases as the multiplex level of the preamplification increases 26

27 Heat Map of 1152 Array 27

28 Comparison for 65 Assays R = Array ΔΔCT ΔΔC T Reference: POLR2A 28

29 Concluding Remarks qpcr is now a >$1 billion dollar industry qpcr arrays dramatically increase throughput at an affordable cost One current challenge is analyzing transcript levels for multiple genes across multiple samples for one or a small number of cells The Matrixed Primer approach shows promise as a method to survey hundreds to thousands of genes in limited samples 29