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

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Cancer Research Challenge: 1. Cell heterogenous gene expression variation 2. Identify and confirm mutations 96

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Active Chemo

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* CTC enumeration determined by castPCR assay #6224 for EGFR mutation p.L858R

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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

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TaqMan® Mutation Detection Assays

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ARMS PCR and Sanger Sequencing

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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

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TaqMan® Mutation Detection Assays

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ARMS PCR and Sanger Sequencing

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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

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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

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Novel approach of multiple analysis from a single cell How to interrogate microRNA, mRNA and protein from a population of single cells

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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”

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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

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Novel approach of multiple analysis from a single cell

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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

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Novel approach of multiple analysis from a single cell

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• 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

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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

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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