Download Understanding the dynamics of gene expression within

Understanding the dynamics of gene
expression within single cell population
Dr. Amy Lam
Application Field Scientist
The world leader in serving science
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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 <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
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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
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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
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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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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”
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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
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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!
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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.
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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.
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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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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
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Detection of somatic cancer mutations using SNP assays
Digital PCR in Quantstudio 12K Flex for Variant Confirmation
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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.
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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%
• Solution: Digital partitioning can be
used to “enrich” for the minor allele
thereby extending the “specificity” of
the assay
1:13
1:1
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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
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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.
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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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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?
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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
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Detection of rare mutations in circulating tumor cells
CastPCR in Mutation Detection Assays with Quantstudio 12K Flex for Rare
Variant Monitoring
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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
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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 )
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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)
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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)
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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
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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
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
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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
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
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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
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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
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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
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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
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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
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