1The world leader in serving science

Dr. Amy LamApplication Field Scientist

Understanding the dynamics of gene expression within single cell population

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Ion Personal Genome Machine (PGM™) for rapid

targeted region sequencing, & expression

analysis

Capillary Electrophoresis for variant confirmation

Real Time PCR Systems for variant ID & rapid

screening of large numbers of samples

TaqMan® Assays for largest catalog of catalog assays and streamlined

custom pipeline

Discovery ApplicationValidation

Genetic Analysis ParadigmDiscovery – Validation - Application

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• Cells are not homogenous

• Cellular heterogeneity may be masked in standard gene expression analysis

Cancer Research Challenge:1. Cell heterogenous gene expression variation2. Identify and confirm mutations <5% frequency 

Strategy for Single Cell Gene Expression Profiling and Rare Mutation Detection are important

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Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established

• Gene Expression ProfilingAnalysis 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 analysisNovel approach of multiple analysis from a single cell

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Quantstudio 12K Flex Real Time PCR System

Gene expression-mRNA, small RNA,

siRNA

Allelic discrimination

Presence / absence

Copy number

Absolute quantification

High-resolution melting

Digital PCR

Protein expression

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Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established

• Gene Expression ProfilingAnalysis 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 analysisNovel approach of multiple analysis from a single cell

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Differential Cell response to HormoneAP-1-bla ME180 EGF Induction

Plate ME180 cells inassay media and growovernight

Replace with assaymedia alone (-EGF)

Replace with assay media containing 10ng/mL EGF

•Incubate for 4hr•Detach and count cells•Stain cells with Live/Deadstain•FACS single cells into 5 five96-well plates/treatment

•Preparation cDNA using Single Cell-to-CT kit

•Use QS12Flex Openarray to analyze 800 single-cell samples and 96 samples of pooled 100-cell/treatment

Analysis of Cellular Heterogeneity from Single-Cell Profiling

From Application note of Life Technologies with title “Analysis of cellular heterogeneity: single-cell profiling on the OpenArray Real time PCR system”

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Single Cell

Reverse Transcription

TaqmanMultiplex

PreAmplification

TaqmanOpenArrayReal-Time

qPCR

Additions to a single tube

Single Cell-to-CT®

Lysis buffer

Use of Single Cell-to-Ct® kit with OpenArray Platform

Analysis of Cellular Heterogeneity from Single-Cell Profiling

1-10cells

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OpenArray® Block User Workflow Overview

The assays are spotted on the OpenArray® plate

Load your samples with Master Mix onto the OpenArray® plate

Cycle and image up to 4 OpenArray® plates (Optional: 9700 Flat block for off line Genotyping)

Results!

TaqMan® assays ordered on-line are sent to Woburn facility

At the Researcher’s Lab

At Life Technologies

Place lid on to array case, load on to carrier

OpenArray® plates staked in case

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The average gene expression may not reflect the variability exhibited by single cells

Existence of two subpopulations of cells expressing EGR1 gene is evident in treated cells. Such heterogeneity is masked in 100-cell pooled sample.

Distribution of Ct among 840 single-cell samples, and insets show distribution among 96 samples of pooled 100-cell samples.

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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.

Analysis of Cellular Heterogeneity from Single-Cell Profiling

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Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established

• Gene Expression ProfilingAnalysis 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 analysisNovel approach of multiple analysis from a single cell

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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 assayA single tube contains•Forward Primer•Reverse Primer•2x TaqMan® probes, one is VIC-labelledand one is FAM-labelled

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Digital PCR in Quantstudio 12K Flex for Variant Confirmation

Detection of somatic cancer mutations using SNP assays

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Digital PCR Overview

Use the number of positive and negative PCR reactions to count the number of target molecules.

Target Quantification

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.

gDNA, cDNA, RNA, plasma

Distribution PCR ReactionPoisson fit and readoutPreparation

Positive reactionsNegative reactions

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• 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

• Solution: Digital partitioning can be used to “enrich” for the minor allele thereby extending the “specificity” of the assay

1:11:13

Mutation Detection: 1%

Detection of somatic cancer mutations using SNP assays

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Standard TaqMan® SNP Genotyping Assays 1% Rare mutation confirmation enabled with digital

Dilution4.5 copies : 445.5 copies 0.1 copies : 9.9 copies

Mutant signal “emerges” at high dilution

Detection of somatic cancer mutations using SNP assays

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• 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.

Detection of somatic cancer mutations using SNP assays

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Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established

• Gene Expression ProfilingAnalysis 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 analysisNovel approach of multiple analysis from a single cell

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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?

Detection of rare mutations in circulating tumor cells

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Two Mega Challenges in CTC Research

Difficult to isolate:• CTCs are rare

• 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

Difficult to characterize:• Definition of CTCs?

• 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

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CastPCR in Mutation Detection Assays with Quantstudio 12K Flex for Rare Variant Monitoring

Detection of rare mutations in circulating tumor cells

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What is the CastPCR• CastPCR refers to competitive

allele-specific TaqMan® PCRwhich is highly specific and capable of finding a needle in a haystack.− Allele-specific primer 1/2 (ASP1/2)− Allele-specific blockers 2/1

(ASB2/1)− Locus-specific primer (LSP)− Locus-specific TaqMan probe

(LST)

• Key characteristics of castPCR− TaqMan® assay-based− Homogenous assays− Highly specific and sensitive− Ease of use; fast (< 2 hr)

A

G

T*

C*

Allele 1 - specific PCR (wild-type)

ASB2

ASP2

LST

LSP

LSP

LST

G

MGB

A

MGB

ASP1

ASB1

* Modified bases

4

1

2 3

Allele 2 - specific PCR (mutant)

MGB = minor groove binder

Detection of rare mutations in circulating tumor cells

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New Idea: RT-qCastPCR for Direct CTC Analysis5 mL blood

Aliquot

Step 2. RNA/DNA Extraction using MagMAX™ Sample Preparation Systems

CK19 etc.

52 μL/wellStep 1. Digital enrichment

(96-fold)

Step 3. RT/Pre-amplification

Step 4. CastPCR & qPCR for CTC enumeration & molecular characterization (2-3-plex readout possibly)

Mutation #1

Mutation #2

( = CTC)

( = Positive ll )

Detection of rare mutations in circulating tumor cells

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Digital RT-qCastPCR Detects Known Lung Cancer Cells Spiked Into Normal Blood Samples

Normal blood samples (uL/well)

RT-qCastPCR for EGFR L850R mutation

RT-qPCR for CK19 mRNA

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Results(No. positive wells) 0 17 - 21

H1975 “CTC” spiked-in (cells/well) 0 ~20

Detection of rare mutations in circulating tumor cells

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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

Detection of rare mutations in circulating tumor cells

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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)

BRAF

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

Detection of rare mutations in circulating tumor cells

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• Circulating tumor cells were successfully detected by RT-qCastPCR in cell spike-in and lung cancer patient samples,demonstrating the power of castPCR for digital CTC counting andmutation 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

Detection of rare mutations in heterogenous population

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Strategy for Single Cell Gene Expression Profiling and Mutation detection via qPCR has been established

• Gene Expression ProfilingAnalysis 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 analysisNovel 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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• 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

Novel approach of multiple analysis from a single cell

From Application note of Life Technologies with title “Novel approach to single cell analysis:multiple analytemeasurements from a single cell”

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• Protein Detection from 10-500 cells or1-1000 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

Detection of protein expression by TaqMan® Protein Assays

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TaqMan® MicroRNA & Small RNA Assays 

Deliver Exceptional Specificity and Sensitivity

• Four Specific Oligonucleotides

1. Looped RT primer2. Forward primer 3. Reverse primer4. TaqMan® probe

1

2

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Detection of microRNA expression by TaqMan® microRNA Assays

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Novel approach of multiple analysis from a single cell

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

Ct

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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

Novel approach of multiple analysis from a single cell

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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

Novel approach of multiple analysis from a single cell

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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

TaqMan® Drug Metabolism Genotyping Assays

TaqMan® Gene Expression AssaysGene expression analysis

Drug metabolism genotyping

MicroRNA & noncoding RNA analysisTaqMan® microRNA AssaysMegaplex microRNA array

Protein expressionTaqMan® Protein Assays

SNP genotypingTaqMan® SNP Genotyping Assays

Copy number variationTaqMan® Copy Number Assays

TaqMan® Gene Signature Plates