ncounter TM Analysis System

Transcription

1 ncounter TM Analysis System Molecules That Count TM

2 Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibilty ncounter Applications to Cancer Analysis

3 Key Milestones in NanoString History

4 Discovery to Dx Discovery Validation Routine testing Whole Genome Gene Expression NanoString NanoString 1-1 s samples 1, s genes 1-1 s samples 1 s 5 genes 1, s -1, s samples 1 s 5 genes

5 Gene Expression Technology Fit NanoString enables sensitive analysis of hundreds of genes QPCR NanoString Sensiti ivity Microarrays Multiplexing Capability (# of Transcripts)

6 ncounter Analysis System Up to 576 genes per rxn Digital Detection and Analysis No Enzymes Required Easy-to-use System Fully Automated Highly Sensitive, Reproducible Data

7 ncounter Analysis System ncounter Prep Station Fully automated sample processing Processes 12 samples in 2 hrs Uses pre-packaged consumables ncounter Digital Analyzer Fully automated data collection Up to 6 cartridges per run 24 hour processing unattended Over 41, data points in 24 hrs

8 Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

9 Probe Architecture Biotin Target Specific Capture Probe Target Specific Reporter Probe Target Specific Capture & Reporter Probes are designed that bind to the mrna transcript

10 Probe Architecture Target Specific Capture Probe Target Specific Reporter Probe Target Specific Capture & Reporter Probes are designed that bind to the mrna transcript

11 ncounter CodeSet Target Specific Capture Probe System Controls Target Specific Reporter Probe Sequence Specific Capture Probes, Reporter Probes and System Controls are Combined to Create Your Custom CodeSet

12 Comprehensive Internal Controls Hybridization controls Positive controls are synthetic RNA targets spiked in at known concentrations (.1fM 1fM) Ensure proper hybridization conditions and no RNase contamination Used as a titration curve to quantitate endogenous transcripts Limit of quantification threshold Negative controls are reporter probe pairs for which no known target should exist within the endogenous sample Used when performing a Student s T-Test to determine presence or absence of an endogenous transcript Purification controls Positive & negative Alignment and immobilization controls

13 ncounter Assay Automated Process Hybridize CodeSet to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes mrna Capture & Reporter Probes

14 ncounter Assay Automated Process ncounter Prep Station Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Hybridized mrna Excess Reporters

15 ncounter Assay Automated Process ncounter Prep Station Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Surface of cartridge is coated with streptavidin Hybridized Probes Bind to Cartridge

16 ncounter Assay Automated Process ncounter Prep Station Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Reporters are aligned and immobilized for imaging and barcode counting

17 ncounter Assay Automated Process ncounter Digital Analyzer Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Image Surface One reporter code = 1 mrna

18 ncounter Assay Automated Process ncounter Digital Analyzer Hybridize Reporter to RNA Remove excess reporters Bind reporter to surface Immobilize and align reporter Image surface Count codes Code Gene Count x 3 y 1 z 2 Codes are counted and tabulated

19 Data Output Simple enough to analyze in Excel TM

20 Simple Protocol Buffer & CodeSet Sample Overnight Solution Phase Hybridization Automated Processing: 12 samples in 2 hr Collect Data: 2 min/sample

21 Validation Study Design Example 35 3 Reaction Number Comparison Calculator Number of genes 1 Number of samples 1 Technical Replicates 3 NanoString qpcr 3 3, No. rxns to complete study NanoString QPCR Validate all the genes of interest to identify the most informative set Interrogate larger sample populations Reduce time to answer Reduce Complexity Without Reducing Content

22 Validation Study Design Example 35 3 Reaction Number Comparison Calculator Number of genes 2 Number of samples 1 Technical Replicates 3 NanoString qpcr 3 6, No. rxns to complete study NanoString QPCR Validate all the genes of interest to identify the most informative set Interrogate larger sample populations Reduce time to answer Reduce Complexity Without Reducing Content

23 Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

24 Assay Reproducibility Replicate e 1 Counts Reproducibility of NanoString Assay Technical Replicates R 2 = Replicate 2 Counts Data Courtesy of Dr. Roger Bumgarner Highly reproducible technical replicates

25 Fractional Fold Change x dow n 67, 15, 2ng Total RNA Counts x up 2x up y = 1.965x R² =.998 y = 1.5x R 2 =.999 y =.676x R 2 = ng Total RNA Counts

26 Fractional Fold Change 2 1,8 1,6 1,4 1,2 1,8,6,4,2 * * * * * * * * * 1fM 1fM 1fM * p<.5 vs

27 Correlation with qpcr Gene 1 Gene 2 Gene 3 Gene 4 Gene EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h Gene 6 Gene 7 Gene 8 Gene 9 Gene EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h Gene 11 Gene 12 Gene 13 Gene 14 Gene EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h Gene 16 Gene 17 Gene 18 Gene 19 Gene EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h EGG 9.3h 18h 24h 33h 48h 7h Data Courtesy of Dr. Eric Davidson 2 genes across 7 time points in quadruplicates: 2 x 7 x 4 = 56 qpcr reactions 7 x 4 = 28 NanoString reactions NanoString QPCR

28 Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

29 Flexibility in Sample Input Total RNA Amplified RNA from Small Amount of Sample Whole Cell Lysates PaxGene Lysed Whole Blood Total RNA Extracted from FFPE Samples

30 Amplified vs. Unamplified Total RNA Log2 Fold Change 5ng BR/HR Amp plified Total RNA R² =,949 8, 6, 4, 2,, -8, -6, -4, -2,, 2, 4, 6, 8, -2, -4, -6, -8, Log2 Fold Change 1ng BR/HR Unamplified Total RNA Human Reference and Brain Reference RNA were used to generate Fold Change Data

31 Cell Lysate and Matched Total RNA Counts in Lysate Counts in Total RNA (1 ng)

32 Lysed vs Purified PaxGene Blood Data Blood Lysate Prep 1 Blood Lysate Prep 2 Lysed Bl lood y =,976x +,337 R² =,983 y =,985x +,451 R² =, Purified Total RNA No globin mitigation

33 FFPE & Frozen vs. Fresh RNA FFPE & Frozen Tissue Derived Total RNA Heart total vs frozen Heart total vs FFPE y =,98x +,13 R² =,928 y =,883x -,48 R² =, Fresh Tissue Derived Total RNA

34 Agenda NanoString Technologies History Introduction to the ncounter Analysis System CodeSet Design and Assay Principals System Performance Sample Type Flexibility ncounter Applications to Cancer Analysis

35 Ewing s Sarcoma Fusion Transcripts Wild-type EWS Wild-type FLI EWS exon 7-FLI exon 5 fusion EWS exon 5-FLI exon 5 fusion EWS exon 7-FLI exon 6 fusion

36 Ewing s Sarcoma Fusion Transcripts B EWS exon 7 capture probe; FLI exon 5 reporter probe B B

37 Ewing s Sarcoma Fusion Transcripts B EWS exon 7 capture probe; FLI exon 6 reporter probe B B

38 Ewing s Sarcoma Fusion Transcripts B EWS exon 7 capture probe; FLI exon 6 reporter probe B B B

39 Ewing s Sarcoma Fusion Transcripts Experiment: Prepare RNA (or lysate) from tumor samples Hybridize to CodeSet designed against specific breakpoints Determine which of the breakpoint-specific probes predominates Prediction: since tumor is likely to be monoclonal, only one of the transcripts will be present at high concentrations

40 Ewing s Sarcoma Fusion Transcripts Transfected cells EWS Cell Line A EWS Cell Line B EWS Cell Line C EWS Cell Line D EWS Cell Line E EWS Cell Line F EWS Tumor A EWS Tumor B FFPE Tumor A FFPE Tumor B 1% Concordance with known breakpoints

41 Ewing s Sarcoma Fusion Transcripts Data courtesy of Marc Ladanyi, MD

42 ncounter Analysis System Very simple & automated assay Direct measurement of target No enzymes; No RT; No amplification Digital data 1 code count = 1 mrna Sensitive, linear, quantitative & reproducible Up to 576 genes measured in a single tube Over 4,32 data points can be generated in 24 hours (72 samples x 56 genes) No sample preparation

43 ncounter TM Analysis System Molecules That Count TM