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Understanding the dynamics of gene expression within single cell population Dr. Amy Lam Application Field Scientist The world leader in serving science 1 Genetic Analysis Paradigm Discovery – Validation - Application Ion Personal Genome Machine (PGM™) for rapid targeted region sequencing, & expression analysis Discovery Capillary Electrophoresis for variant confirmation Real Time PCR Systems for variant ID & rapid screening of large numbers of samples Validation TaqMan® Assays for largest catalog of catalog assays and streamlined custom pipeline Application 2 Cancer Research Challenge: 1. Cell heterogenous gene expression variation 2. Identify and confirm mutations <5% frequency • Cells are not homogenous • Cellular heterogeneity may be masked in standard gene expression analysis Strategy for Single Cell Gene Expression Profiling and Rare 3 Mutation Detection are important Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established • Gene Expression Profiling Analysis of cellular heterogeneity from single-cell profiling • Mutation Detection (A) Detection of somatic mutation in heterogenous population (B) Detection of rare event in circulating tumor cells • Multiple analysis Novel approach of multiple analysis from a single cell 4 Quantstudio 12K Flex Real Time PCR System Gene expressionmRNA, small RNA, siRNA Protein expression Allelic discrimination Presence / absence Digital PCR High-resolution melting Copy number Absolute quantification 5 Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established • Gene Expression Profiling Analysis of cellular heterogeneity from single-cell profiling • Mutation Detection (A) Detection of somatic mutation in heterogenous population (B) Detection of rare event in circulating tumor cells • Multiple analysis Novel approach of multiple analysis from a single cell 6 Analysis of Cellular Heterogeneity from Single-Cell Profiling Differential Cell response to Hormone AP-1-bla ME180 EGF Induction Replace with assay media alone (-EGF) •Incubate for 4hr •Detach and count cells •Stain cells with Live/Dead stain •FACS single cells into 5 five 96-well plates/treatment •Preparation cDNA using Single Cell-to-CT kit Plate ME180 cells in assay media and grow overnight Replace with assay media containing 10ng/mL EGF •Use QS12Flex Openarray to analyze 800 single-cell samples and 96 samples of pooled 100-cell/treatment From Application note of Life Technologies with title “Analysis of cellular heterogeneity: single-cell profiling on the OpenArray Real time PCR system” 7 Analysis of Cellular Heterogeneity from Single-Cell Profiling Use of Single Cell-to-Ct® kit with OpenArray Platform Additions to a single tube Single Cellto-CT® Lysis buffer Single Cell Reverse Transcription Taqman Multiplex PreAmplification Taqman OpenArray Real-Time qPCR 1-10cells 8 OpenArray® Block User Workflow Overview At Life Technologies TaqMan® assays ordered online are sent to Woburn facility The assays are spotted on the OpenArray® plate OpenArray® plates staked in case At the Researcher’s Lab Load your samples with Master Mix onto the OpenArray® plate Place lid on to array case, load on to carrier Cycle and image up to 4 OpenArray® plates (Optional: 9700 Flat block for off line Genotyping) Results! 9 The average gene expression may not reflect the variability exhibited by single cells Distribution of Ct among 840 single-cell samples, and insets show distribution among 96 samples of pooled 100-cell samples. Existence of two subpopulations of cells expressing EGR1 gene is evident in treated cells. Such heterogeneity is masked in 100-cell pooled sample. 10 Analysis of Cellular Heterogeneity from Single-Cell Profiling The use of Single Cell-to-Ct kit. TaqMan assays together with OpenArray platform 1. To provide an effective solution for quickly profiling a large number of cells across a panel of 56 to 244 genes 2. to evaluate a greater number of single-cell events in order to achieve statistical significance. 11 Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established • Gene Expression Profiling Analysis of cellular heterogeneity from single-cell profiling • Mutation Detection (A) Detection of somatic mutation in heterogenous population (B) Detection of rare event in circulating tumor cells • Multiple analysis Novel approach of multiple analysis from a single cell 12 Detection of somatic cancer mutations using SNP assays • Problem: Standard TaqMan® SNP Genotyping Assays are challenged in detecting minor alleles at less than (30%) ~1:3. Cancer researchers are interested in detecting somatic mutations at 1% or less Mutation Detection: 1% TaqMan SNP assay A single tube contains •Forward Primer •Reverse Primer •2x TaqMan® probes, one is VIC-labelled and one is FAM-labelled 13 Detection of somatic cancer mutations using SNP assays Digital PCR in Quantstudio 12K Flex for Variant Confirmation 14 Digital PCR Overview Digital PCR is an analytical technique for quantification of nucleic acid samples based on PCR amplification of single template molecules, without reference to a standard curve. Preparation gDNA, cDNA, RNA, plasma Distribution PCR Reaction Positive reactions Negative reactions Poisson fit and readout Target Quantification Use the number of positive and negative PCR reactions to count the number of target molecules. 15 Detection of somatic cancer mutations using SNP assays • Problem: Standard TaqMan® SNP Genotyping Assays are challenged in detecting minor alleles at less than (30%) ~1:3. Cancer researchers are interested in detecting somatic mutations at 1% or less Mutation Detection: 1% • Solution: Digital partitioning can be used to “enrich” for the minor allele thereby extending the “specificity” of the assay 1:13 1:1 16 Detection of somatic cancer mutations using SNP assays Standard TaqMan® SNP Genotyping Assays 1% Rare mutation confirmation enabled with digital Dilution 4.5 copies : 445.5 copies 18000 18000 16000 16000 14000 14000 12000 12000 10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0 0 0 1000 2000 3000 4000 5000 0.1 copies : 9.9 copies Mutant signal “emerges” at high dilution 0 1000 2000 3000 4000 5000 17 Detection of somatic cancer mutations using SNP assays • Rare mutation can be detected by SNP assay with digital PCR in real time PCR platform, allowing the accurate allelic gene expression analysis in heterogeneous population. • Digital partitioning can be used to “enrich” for the minor allele thereby extending the “specificity” of the assay, down to 1% mutation detection. 18 Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established • Gene Expression Profiling Analysis of cellular heterogeneity from single-cell profiling • Mutation Detection (A) Detection of somatic mutation in heterogenous population (B) Detection of rare event in circulating tumor cells • Multiple analysis Novel approach of multiple analysis from a single cell 19 Detection of rare mutations in circulating tumor cells What are Circulating Tumor Cells (CTCs)? • A definition (according to CellSearch developers) • EpCAM positive • CK8, 18 or 19 positive • CD45 negative • Intracellular nucleus • ≥ 4 × 4 µm in size • Other specific morphological features • Tumor-specific mutation(s) or genomic amplifications? 20 Two Mega Challenges in CTC Research Difficult to isolate: Difficult to characterize: • CTCs are rare • Definition of CTCs? • Few CTCs in 1 mL blood • High background of normal blood cells (~107 cells / mL blood) • CTCs are fragile in vitro • Cell apoptosis • Cell lysis • CTCs are difficult to capture • • • • Lack of robust markers Poor affinity Not so specific Take long time • There are few CTCs available • Few CTCs in 1 mL blood • CTCs are not pure • • • • Normal cell contamination Heterogeneity of tumors (EpCAM etc.) De novo mutation Presence of circulating tumor DNA (CTD) • Lack of tools for mol characterization • • • • • Single cell analysis capability Multiplexed Reproducible Fast to results Lower cost 21 Detection of rare mutations in circulating tumor cells CastPCR in Mutation Detection Assays with Quantstudio 12K Flex for Rare Variant Monitoring 22 Detection of rare mutations in circulating tumor cells What is the CastPCR • CastPCR refers to competitive TaqMan® allele-specific PCR which is highly specific and capable of finding a needle in a haystack. Allele 1 - specific PCR (wild-type) 2 ASP1 − MGB 1 4 LSP Allele 2 - specific PCR (mutant) ASP2 TaqMan® assay-based − Homogenous assays − Highly specific and sensitive − Ease of use; fast (< 2 hr) LST G − Allele-specific blockers 2/1 • Key characteristics of castPCR T* A ASB2 − Allele-specific primer 1/2 (ASP1/2) (ASB2/1) − Locus-specific primer (LSP) − Locus-specific TaqMan probe (LST) 3 C* G LST LSP ASB1 MGB A * Modified bases MGB = minor groove binder 23 Detection of rare mutations in circulating tumor cells New Idea: RT-qCastPCR for Direct CTC Analysis 5 mL blood Aliquot 52 μL/well ( = CTC) Step 1. Digital enrichment (96-fold) Step 2. RNA/DNA Extraction using MagMAX™ Sample Preparation Systems Step 3. RT/Pre-amplification CK19 etc. Mutation #1 Mutation #2 Step 4. CastPCR & qPCR for CTC enumeration & molecular characterization (2-3plex readout possibly) ( = Positive ll ) 24 Detection of rare mutations in circulating tumor cells Digital RT-qCastPCR Detects Known Lung Cancer Cells Spiked Into Normal Blood Samples Normal blood samples (uL/well) 52 52 H1975 “CTC” spikedin (cells/well) 0 ~20 0 17 - 21 RT-qPCR for CK19 mRNA RT-qCastPCR for EGFR L850R mutation Results (No. positive wells) 25 Detection of rare mutations in circulating tumor cells Detection of Circulating Tumor Cells (CTCs) in Lung Cancer Patients by RT-qCastPCR Case No. Age (Yrs) Stage Treatment Status *CTC Counts/mL 1 86 IB Pre-Treatment 11 2 63 IB Pre-Treatment 11 3 79 IIB Pre-Treatment 32 4 75 IV Active Chemo >96 5 71 IV Active Chemo >96 * CTC enumeration determined by castPCR assay #6224 for EGFR mutation p.L858R 26 Detection of rare mutations in circulating tumor cells Detection of Mutations in Heterogeneous Tumor Research Samples by TaqMan® Mutation Detection Assays Study 1: KRAS mutation in colorectal tumor FFPE research samples (n=51) G12A G12C G12D G12R G12S G12V G13D WT TaqMan® Mutation Detection Assays 2 2 12 0 0 4 11 30 ARMS PCR and Sanger Sequencing 2 2 12 0 0 4 11 30 Study 2: BRAF, KRAS and EGFR mutations in cell Line, FFPE and fresh frozen tumor research samples (n=33) BRA F EGFR T790M EGFR L858R EGFR G719D EGFR Del E746-A750 (6223) EGFR Del E746-A749 (6225) 7 KRAS mutations WT TaqMan® Mutation Detection Assays 1 1 3 1 2 3 18 6 ARMS PCR and Sanger Sequencing 1 1 2 1 2 3 17 7 27 Detection of rare mutations in heterogenous population • Circulating tumor cells were successfully detected by RTqCastPCR in cell spike-in and lung cancer patient samples, demonstrating the power of castPCR for digital CTC counting and mutation characterization directly from blood • Data Performance • High sensitivity: 1-5 copies • TaqMan® workflow: simple, fast, and homogenous detection • Key applications • Mutation analysis of tumor s • Direct CTC analysis • Prenatal analysis • Allelic gene expression analysis 28 Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established • Gene Expression Profiling Analysis of cellular heterogeneity from single-cell profiling • Mutation Detection (A) Detection of somatic mutation in heterogenous population (B) Detection of rare event in circulating tumor cells • Multiple analysis Novel approach of multiple analysis from a single cell 29 Novel approach of multiple analysis from a single cell How to interrogate microRNA, mRNA and protein from a population of single cells 30 Novel approach of multiple analysis from a single cell • Human fibrosarcoma cells lines (HT1080) were transfected with the expression vector FUS-GFP, which encodes a GFP-tagged human FUS (fused in sarcoma) protein. • FUS gene encodes a multifunctional protein that regulates gene activity through interacting with other proteins, RNAs, and DNA. • Individual cells expressing GFP were isolated by FACS • Protein expression detected by TaqMan Protein assay • MicroRNA expression is detected by TaqMan microRNA assay • mRNA expression is detected by TaqMan Gene Expression assay From Application note of Life Technologies with title “Novel approach to single cell analysis:multiple analyte measurements from a single cell” 31 Detection of protein expression by TaqMan® Protein Assays • Protein Detection from 10-500 cells or 1-1000 ng protein from tissues • Combines protein detection using Antibodies with robust, sensitive real-time PCR • Relative quantification of proteins in cell and tissue lysates • no purification of proteins required! • Just lyse and dilute…. • Utilize Proximity Ligation Assay Technology • Applications: − − Small sample protein analysis Correlation of RNA & Protein > miRNA:protein > mRNA:protein − − − − Validation of siRNA induced silencing Validation of Gene Transfection/Transduction Sample analysis from FFPE & Frozen Tumor tissues Analysis of in vitro protein:protein interactions 32 Detection of microRNA expression by TaqMan® microRNA Assays TaqMan® MicroRNA & Small RNA Assays Deliver Exceptional Specificity and Sensitivity 1 • Four Specific Oligonucleotides 1. Looped RT primer 2. Forward primer 3. Reverse primer 4. TaqMan® probe 2 4 3 33 Novel approach of multiple analysis from a single cell Ct The protein in single cells measured by PLA-qPCR correlated with GLP fluorescence measured by FACS in the same cell. The FUS-GFP protein production was varied from every single cells 34 Novel approach of multiple analysis from a single cell Ct Ct Ct Ct Ct Ct Ct • Differences in transcription and translation efficiencies in individual cells •Strong correlation between the number of FUS molecules and FUS-GFP mRNA •TaqMan Assays are measuring true biological events 35 Novel approach of multiple analysis from a single cell Use of TaqMan assays • Provide a simple workflow with minimal analyte loss from a single cell • Enable measurement of protein, nucleic acids in a single cell population, having the correlation studies of cellular response in protein/DNA/mRNA/microRNA 36 Conclusion Openarray platform or real time PCR platform with TaqMan assays provide • Validated workflow for absolute quantification and global gene profiling in single cells •Validated workflow to measure multiple types of analytes in a single cell •Validated workflow to detect rare mutation in a heterogeneous population 37 Products Overview Gene expression analysis TaqMan® Gene Expression Assays TaqMan® Gene Signature Plates Drug metabolism genotyping TaqMan® Drug Metabolism Genotyping Assays MicroRNA & noncoding RNA analysis TaqMan® microRNA Assays Megaplex microRNA array Protein expression TaqMan® Protein Assays SNP genotyping TaqMan® SNP Genotyping Assays Copy number variation TaqMan® Copy Number Assays 38