using flow cytometry to speed determination of eukaryotic genome sizes and cell type-specific gene...
TRANSCRIPT
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Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Broadcast Date: Wednesday, February 1, 2012
Time: 1:00 pm EST, 10:00 am PST
Sponsored by
Using Flow Cytometry to Speed Determination of
Eukaryotic Genome Sizes and Cell Type-Specific
Gene Expression
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Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Your Moderator
Tamlyn Oliver Managing Editor
Genetic Engineering & Biotechnology News
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Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
David Galbraith, Ph.D. Professor
BIO5 Institute and School of Plant Sciences
University of Arizona
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Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression
Technical aspects of flow analysis and sorting in plants
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http://cba.musc.edu/flowcytometry
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Dealing with Multicellular Tissues in Flow Cytometry
• How to fit a plant through a flow tip?
70 μm
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B A
C D
Make protoplasts (single cells lacking a cell wall)
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DNA flow histogram of fixed tobacco leaf protoplasts. Poor CV due to uneven illumination within the flow stream.
CRBC
CV ~7-10%
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DNA flow histogram of tobacco nuclei released from lysed leaf protoplasts. Improved CV since nuclei are more evenly illuminated.
CRBC
CV ~3.5%
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Problem with this approach: • Protoplasts are not easy to prepare. Some species and
organs cannot be made into protoplasts.
Simple solution: release nuclei by “chopping”
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Method for DNA content and ploidy estimation 1. Select tissue of interest. 2. Place tissue in petri dish, in cold “chopping” medium. 3. Chop tissue using a single-edge razor blade, for approximately 1 min. 4. Filter tissue through nylon mesh (pore size 15-50 μm). 5. Add appropriate fluorochrome to desired concentration. 6. Analyze fluorescence emission using flow cytometry.
Galbraith et al., Science 220:1049-1052 (1983)
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A. DNA flow histogram of a chopped tobacco leaf.
B. DNA flow histogram of a chopped arabidopsis leaf.
B
CV ~3.5%
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Applications of the chopping technology
Genome size measurements for essentially all plant species and tissue/organ types. Characterization of natural and agricultural populations: ploidy screening and identification of distributions. Ecology and crop improvement. Identification of aneuploidy. Characterization of unsuspected phenomena (for example, somatic endoreduplication). Molecular and cellular biology of the nucleus (more later).
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DAPI Fluorescence (log)
0 200 400 600 800
Num
ber
of N
ucle
i
0
1000
2000
3000
4000
Chopping can be used with animal tissues and organs
This includes insects and nematodes, as well as mammalian species.
Brain
nuclei
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Extending genome size observations to the entire angiosperms
We estimate about 500,000 species of flowering plants in the world. Approximately 15,000 species are becoming extinct per year due to anthropogenic change. Some of these will be plant species, and some will be as yet undescribed. Approximately 2% of known species have a minimal basal molecular description. There is a need for a global molecular census of the angiosperms.
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A global inventory of flowering plants
Start with Genome Size Measurements: Fundamental to our understanding of eukaryotic species. Limited information available in the RBG Kew C-value database (~1-2% of angiosperms). Flow cytometry is ideal for measurements of this type, and can be extended to all 600,000 species. Problem of large dynamic range of C values.
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Gregory TR (2005). Ann. Bot. 95:133-146.
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The Accuri C6 Portable Flow Cytometer
• Two lasers. • No user adjustments. • 24-bit ADC. • No pressurized sheath.
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Dynamic range of the Accuri flow
cytometer (1.9 x 107 bins) is greater
than this range of DNA content values
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FL2-A (x 10-5)
0 5 10 15
Nu
mb
er
of N
ucle
i
0
200
400
600
800
FL2-A
102 103 104 105 106
FL
3-A
103
104
105
106
P (1.8%)
A B
FIGURE 1
Gated on Region P
Pisum sativum
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FL2-A
103 104 105 106
FL3-A
105
106
B
FL2-A
103 104 105 106
FL3-A
105
106
P1 1.8%
P1 1.2%
FL2-A
102 103 104 105 106
103
104
105
106
A
FL2-A
Nu
mb
er
of n
ucle
i
0
250
500
750
104
105
C
E
FL2-A
102 103 104 105 106
FL3-A
103
104
105
106
D
104
105
Gated on P1
FL2-AN
um
be
r o
f n
ucle
i
0
250
500
750
F
Gated on P1
FIGURE 2
R1
R1
Arabidopsis thaliana
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FL2-A
104 105 106 107
FL3-A
104
105
106
107
B
P2 4.3%
FL2-A
102 103 104 105 106
103
104
105
106
A
FL2-A (x 10-6) FL2-A
105 106
2C
DN
A C
onte
nt (p
g)
2C
DN
A C
onte
nt (p
g)
FL3-A
D Er2 = 0.999r
2 = 0.999
FIGURE 3
0.0 1.0 2.0 3.0
0
20
40
60
80
FL2-A
Num
ber
of nucle
i
0
250
500
750
104
105
CGated on P2
106
107
At (2C)
At (4C)
At (8C)
Ps (2C)
Ta (2C)
Aa (2C)
R2
At (2C-8C) Ps (2C)
Ta (2C)
Aa (2C)
At (2C)
At (4C)
Ps (2C)
At (8C)
Ta (2C)
Aa (2C)
1
10
100 At: Arabidopsis thaliana
Ps: Pisum sativum
Ta: Triticum aestevum
Aa: Alstroemeria aurea
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• The smallest reported angiosperm genome size is that of Genlisea margaretae (Lentibulariaceae), having a 2C value of 0.129 pg. The largest currently measured is that of Paris japonica (Melanthiaceae) with a 2C value of 304.46 pg, representing a genome of ~150 billion base pairs.
• This is still smaller than the dynamic range measurable using the Accuri C6.
Spanning the Angiosperms
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• ~500,000 species.
• 30 machines.
• 8 samples / hr; 40 hrs/week
• Only 52 weeks total time required for analysis of all species…
• Collection and availability of local taxonomic expertise will
clearly be problems.
Throughput and Time-Frame
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Where does this lead us?
• Availability of the C6 means we are no longer limited in our technological ability to access a complete description of genome sizes for flowering plants.
• Simple technologies (GPS-enabled cell phones with cameras) allows linking of samples and locations.
• This would be followed by NextGen and Gen3 sequencing to describe each of these samples, starting at low coverage.
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Flow Sorting in Plants Given the ability to make suspensions of nuclei, we can now think about experiments involving sorting.
• Analysis of cell type-specific gene expression.
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Nuclear Genome
mRNA
Translation
Degradation
Stages in Gene Expression
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Poly A+ RNA sampling
mRNA
Translation
Degradation
Nuclear Genome
Analysis of gene expression is typically done by sampling total cellular polyA+ RNA, which estimates the steady state concentration within the cell.
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We are looking at transcripts in the nucleus of specific cell types
(Nuclear Transcriptomics) • Zhang et al. (2005): Cell type-specific
characterization of nuclear DNA contents within complex tissues and organs. Plant Methods 2005, 1:7 doi:10.1186/1746-4811-1-7.
• Zhang et al. (2008). Characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiology 147:30-40.
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HTA6 GFP
Strategy:
Nuclear GFP targeting achieved by fusing GFP to histones.
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pSUL2-1::HTA6:GFP (phloem CCs)
pSCR::HTA6:GFP (endodermis)
Cell type-specific labeling of nuclei
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Wild Type
2C 4C 8C 16C
DNA
content
GF
P flu
ore
scence
p35S
GF
P flu
ore
scence
DNA
content
GF
P flu
ore
scence
p35S
The C6 is very
useful for
identification of
suitable transgenic
plants, since
screening is very
rapid.
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Col-0
pRPL16B
pSCR
A B
C
E F
p35S
pSultr 2-1
pSHR D
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Transcriptomics using sorted nuclei
A
C
B
D
E
• Transcripts were amplified from GFP-positive nuclei sorted from phloem companion cell homogenates
• Microarrays were hybridized and genes identified whose transcripts were up (0.2% of total) by factors of two-fold or more (P-value < 0.01).
• We selected the top 12 genes, and employed the 5’-regions for regulating GFP expression in transgenic plants.
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A
C
B
D
E • All 12 showed phloem- or vascular-
specific GFP expression.
Transcriptomics using sorted nuclei
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NPCC4 NPCC3
NPCC6 NPCC5
NPCC2 NPCC1 NPCC7
NPCC12
NPCC10 NPCC9
NPCC11
NPCC8
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How does this compare to “conventional” transcriptomics?
Transcript levels estimated from nuclei are broadly concordant with those estimated from protoplasts. Our results using nuclei agree with those of other groups using sorted protoplasts.
Birnbaum et al. (2003). Science 302:1956-1960.
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Is this approach applicable to animal systems? Flow sorting of specific cell types, labeled through FP expression is a widely used technology. Identification of different cell types in complex tissues can be simple, but preparation of single cells can be very difficult (cf. the CNS). Sorting nuclei would get around this problem, and we can use histone-GFP fusions to label these nuclei.
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(1) Confocal image of live HeLa cells constitutively expressing Histone 2B–GFP. GFP fluorescence (green) was overlaid onto a differential interference contrast image. H2B-GFP is detected in cells at all phases of the cell cycle. H2B-GFP is contained solely in the nucleus.
Image courtesy K. Sullivan
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Does the spectrum of RNA transcripts of animal nuclei correlate with that of the cytoplasm?
Barthelson et al. (2007).
HepG2 cells grown in culture. Analysis and FACS sorting of DAPI-stained nuclei. Isolation of polyA+ RNA. Comparison of expression profiles between cells and nuclei using microarrays.
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Expression profiling using mammalian nuclei
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Conclusions • The nucleus is a valuable source of important
information in eukaryotic organisms.
• The BD Accuri C6 flow cytometer provides unique features that make it particularly suitable for analyzing nuclei.
• The type of information that can be obtained using this instrument will be of profound significance in the future of scientific research in this area.
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Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression
Technical aspects of flow analysis and sorting in plants
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Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Chuck Cannon, Ph.D. Associate Professor
Texas Tech University
Professor, Xishuangbanna Tropical Botanic Garden
Chinese Academy of Sciences
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Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Clare Rogers Senior Marketing Applications Specialist
BD Biosciences-Cell Analysis
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
The BD Accuri® C6 flow cytometer
• Overview of the system
• Ease of use attributes
• DNA analysis-specific attributes
• QC tools for DNA analysis
• Data Analysis
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The BD Accuri C6 flow cytometer
An affordable, full-featured, easy-to-use flow cytometer
Two lasers and six detectors
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The BD Accuri C6 flow cytometer
Innovations in all the major components of a flow cytometer
• Fluidics: Peristaltic pumps and pulse dampeners allow miniaturization
and direct-volume measurement
• Optics: locked-down alignment
• Signal detection: broad dynamic range obviates voltage adjustments
• Software: developed by “high tech anthropologists” trained to facilitate
human-computer interactions
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Fluidics
• Laminar flow fluidics
• Non-pressurized, peristaltic pump-
driven system
• Patented pulse dampeners
• User controls both flow rate and core
diameter
• Volume measurement for absolute
counts
• Minimum sample volume 50 µL if using
a 1.5 mL conical tube
• Up to 10,000 events/second
Sheath
Purge
Waste
Flow Cell Laser
Sa
mp
le
Dete
cto
r
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FSC
SSC
488 nm
solid state laser
640 nm diode laser
PMTs for fluorescence
Detection
Diodes for scatter
detection
Compact optical system
design reduces cost and
eliminates alignment issues
510/15
540/20
565/20
610/20
780/60
3 blue 1 red
2 blue 2 red
4 blue
User changeable
optical filters
Selectable Lasers
Alignment and signal detection are optimized and
locked down at manufacture
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Instrument-to-instrument variation is minimal
8-peak data, multiple C6 instruments manufactured over a six-
month period.
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Instrument set-up is simplified
• Power on
• Water – Bleach – Water
• Validate with beads
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
High Tech Anthropology: Menlo Innovations
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National Oceanic and Atmospheric Administration:
Great Lakes Microcystis research project – Lake Erie
The Ecosystem Centre:
Palmer Peninsula, Antarctica
A Robust and Portable Tool for Challenging Environments
2nd Norwich Flow Day:
Institute for Food Research
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD Accuri C6 functionalities for DNA analysis
• Particle rate control: adjustable fluidics
• Threshold on any parameter: light scatter or fluorescence
o Primary and secondary thresholds allowed
• Area versus Height for aggregate exclusion (all parameters)
• Linear response across the broad dynamic range
• Virtual Gain tool to compensate for sample-to-sample
staining variation
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD DNA QC Particles kit for performance validation
The chicken erythroid nuclei (CEN) preparation will contain singlets,
doublets, triplets, and other aggregates which allow one to check
instrument linearity and resolution.
Singlet Peak
CV = 2.5%
PI FL2-A
CEN
2.0
3.0
4.0 4.9
5.9
6.9
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD DNA QC Particles kit for performance validation
The calf thymus nuclei (CTN) preparation allows one to check the doublet
discrimination ability of the C6 using an Area versus Height plot and to check
resolution for a cycling population
PI FL2-A
PI F
L2-H
Singlet gating
PI FL2-A
CTN
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ModFit LT™
as well
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
FCS Express:
Multicycle FlowJo
Original
BD Accuri
Data
Popular cell cycle analysis software packages can
analyze BD Accuri data files
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Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Q&A
![Page 77: Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression](https://reader033.vdocument.in/reader033/viewer/2022052411/5572106d497959fc0b8d2785/html5/thumbnails/77.jpg)
Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Your Moderator
Tamlyn Oliver Managing Editor
Genetic Engineering & Biotechnology News
![Page 78: Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression](https://reader033.vdocument.in/reader033/viewer/2022052411/5572106d497959fc0b8d2785/html5/thumbnails/78.jpg)
Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
David Galbraith, Ph.D. Professor
BIO5 Institute and School of Plant Sciences
University of Arizona
![Page 79: Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression](https://reader033.vdocument.in/reader033/viewer/2022052411/5572106d497959fc0b8d2785/html5/thumbnails/79.jpg)
Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Chuck Cannon, Ph.D. Associate Professor
Texas Tech University
Professor, Xishuangbanna Tropical Botanic Garden
Chinese Academy of Sciences
![Page 80: Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression](https://reader033.vdocument.in/reader033/viewer/2022052411/5572106d497959fc0b8d2785/html5/thumbnails/80.jpg)
Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Clare Rogers Senior Marketing Applications Specialist
BD Biosciences-Cell Analysis
![Page 81: Using Flow Cytometry to Speed Determination of Eukaryotic Genome Sizes and Cell Type-Specific Gene Expression](https://reader033.vdocument.in/reader033/viewer/2022052411/5572106d497959fc0b8d2785/html5/thumbnails/81.jpg)
Using Flow Cytometry to Speed Determination of Eukaryotic
Genome Sizes and Cell Type-Specific Gene Expression
Thank You For Attending
Using Flow Cytometry to Speed Determination of
Eukaryotic Genome Sizes and Cell Type-Specific
Gene Expression
Broadcast Date: Wednesday, February 1, 2012
Time: 1:00 pm EST, 10:00 am PST
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