NANOPROBE FOR LIVE CELL GENOMICS - Calit2@UCI Symposium...“Targeted Profiling of Breast Cancer...
Transcript of NANOPROBE FOR LIVE CELL GENOMICS - Calit2@UCI Symposium...“Targeted Profiling of Breast Cancer...
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NANOPROBE FOR LIVE CELL GENOMICS
Kumar Wickramasinghe
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COLLABORATORS
• Kavita Arora, PhD –
• Rahul Warrior, PhD –
• Ed Nelson, MD –
• Arthur Lander, MD, PhD –
Development and Cell Biology, UCI
Development and Cell Biology, UCI
Chief, Hematology and Oncology, UCI
Development and Cell Biology, UCICenter for Complex Biological Systems
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We introduce a unique technology that can rapidly profile gene expression levels within a living cell at unprecedented sensitivity - down to single molecules.
Capability opens up
New methods for diagnosis of genetic diseases
New analysis instrument in cell biology for tracking the fate of single cells in response to external stimuli such as drugs, silencing or signaling molecules
New analytical tool that is likely to have significant impact in cell biology
OVERVIEW
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Current technology uses DNA arrays to profile gene expression levels
DNA arrays typically require mRNA extracted from thousands to millions of cells for analysis
Averaging over thousands of cells reduces sensitivity and masks subtle variations in gene expression
The ability to detect low level fluctuations in gene expression in living cells – down to single molecules – is key to detecting genetic mutations that result in diseases and for studying the effects of drugs or signaling molecules
NEEDS THAT ARE NOT MET BY CURRENT TECHNOLOGY
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STOCHASTIC TRANSCRIPTION
Average accurately represents the population
Average hides information about the population
Cells randomly express high or low levels
C
Adapted from: MacNeil L T , Walhout A J Genome Res. 2011
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STOCHASTIC TRANSCRIPTION AND PHENOTYPE
Raser and O’Shea, 2005
C
Fingerprints from identical twins
Genetically identical cloned cats
Stochastic distribution of cell fates in insect photoreceptors
Losick and Desplan, 2008
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object
BACKGROUND OF THE TECHNOLOGY
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Force – Vibrating Mode Atomic Force Microscope - AFM
Light – Apertureless Near-Field - sSNOM, ANSOM, TERS
Heat – Scanning Thermal Microscope - SThM
Magnetism – Scanning Magnetic Force Microscope - MFM
Electrostatics – Electrostatic Force Microscope - EFM
Electrochemical Potential – Scanning Kelvin Probe Microscope - SKPM
Photovoltage – Scanning Photovoltage Microscopy - SPVM
SCANNING PROBE MICROSCOPYNanoscale Measurements of…..
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AFM MOLECULAR SORTING AND DELIVERY(Unal, Frommer, Wickramasinghe, Appl. Phys. Lett. 88, 183105, 2006)
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SCHEMATIC FOR MOLECULAR SAMPLING WITHIN A SINGLE CELL
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~V
Nanoprobe tip modification mRNA extraction from a cellby dielectrophoretic force
Selective mRNA hybridization
mRNA collectionmRNA quantification
Direct and selective sampling of mRNA from a single cell or an array of cellsusing a nanoscale probeNo laborious and expensive chemical purification steps necessarymRNA is copied into cDNA and then quantified using standard qPCR
PLATFORM CONCEPT
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PROOF OF CONCEPTmRNA from a single living cell has been selectively extracted and quantifiedCopy numbers down to a few molecules has been detected for selected mRNA species (HPRT)Multiple mRNA species have been detected at the same timeTransfected breast cancer genes were detected through expression analysis in single cells
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10- fold dilutions ofGAPDH mRNAHPRT mRNA
“Targeted Profiling of Breast Cancer Gene in a Single Living Cell”, Analytical Biochemistry, 2010
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Time (mins)
Ct (
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miRNA quantification of hsa-mir-125a during apoptosis
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Breast cancer gene (rat neu -ve)
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Breast cancer gene (rat neu +ve)
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EPIGENETIC CHANGES CONTRIBUTE TO CANCER GENE MISEXPRESSION
OFF ON
Zhou et al (2011), 12; 7‐18
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1.Testis2. PCR negative control3. Electrophoresis ON4. Electrophoresis OFF5. Whole cell skewer6. cDNA water control
1 2 3 4 5 6Tubulin 5
1 2 3 4 5 6BRCA1
DETECTION OF EPIGENETIC ALLELE SPECIFIC EXPRESSION
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• Large single cell - 0.5 mm long • Can be manipulated under non-sterile conditions• Robust system for microinjections• Rapid development, 6000 cells in 2 hours, larva hatches in 24 hours• Sensitive phenotypic assay for effects on development and viability • Maternal mRNA pool not replenished during development
DROSOPHILA DEVELOPMENT IS A POWERFUL IN VIVO ASSAY
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Sophisticated transgenic tools that allow dynamic visualization of GFP-tagged bcd mRNA in vivoExtensive array of genetic mutations, gene duplications, etc., to aid in calibration and quantitationGenomic data from the ModEncode project
Tracking mRNA Expression in Space and Time
a b c
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CapabilityOur technology
Microfluidics technology
Live cell transcriptomics Yes NoReagent volume 1 ul 50 ulDisease detection Yes NoIn situ pathology Yes NoSpeed 10 secs 1-2 hrsComplexity Simple ComplicatedCost Lower HigherChemical purification steps No YesProtein assays Yes NoSingle cell transfection Yes NoLive cell response Yes No
COMPARISON WITH EMERGING TECHNOLOGIES
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Electric Field ONmRNA collection
Electric Field OFFmRNA DEPOSITION
WHERE WE WANT TO BE ……
semi-automated mRNA extraction platform
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BACKUP
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NorthernMicroarrayReal-time PCRRNA-seq
FISH
MS2-GFP
SCA
Require averaging of gene expression over many cells
Can follow expression in vivo, but requires genetic modification, challenging to quantitate
Can analyze individual cells, but is not dynamic
In vivo, quantitative, single cell analysis, that can be followed over time
COMMON METHODS FOR RNA QUANTIFICATION
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TRANSCRIPTIONAL PROFILING OF CANCER CELLS
Gunn, Nelson, Journal of Biomedical Materials Research, 2010
(c)
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DIFFERENTIAL mRNA LOCALIZATION IN NEURONS
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A B
DISRUPTION OF mRNA LOCALIZATION IN NEURODEGENERATIVE DISEASES
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Detect and monitor transcriptional responses at the single cell level over time
Determine operating parameters relating to sensitivity, spatial resolution, and number of different transcripts that can be assayed simultaneously
Develop methods to maximize sensitivity without affecting the developmental program
Calibration of mRNA extraction procedure
Develop an SCA based system for delivery of siRNAs and proteins for single cell analysis
BROAD GOALS