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1 12/23/2011 | Life Technologies™ Proprietary and confidential
Analyzing Expression Profiles from
Single Stem Cells Using the Single Cell-
to-Ct™ kit
Ron Abruzzese, Ph.D.
Life Technologies
Austin, Texas
2 12/23/2011 | Life Technologies™ Proprietary and confidential| Life Technologies |
Why Study of Single Cells?
Cells are not homogenous
Research into rare cell
or event
• Circulating metastatic cells
• Fetal cell in maternal blood
• Event within a library
Research using
scarce, precious
sample
• Archival tissue (FFPE)
• Clinical sample (fresh tumor)
• Biomarker discovery
•Data from ensemble averaging in cell
measurements can be misleading
• Interesting Biological Research Applications
Learn more about the
Single Cell-CT Kit (400 Reactions)
3 12/23/2011 | Life Technologies™ Proprietary and confidential
Develop a complete workflow to obtain
statistically relevant single cell data
Dynabeads®
CD3/CD28
Naive or
resting
T cell
Benefits
•Optimized reagents
•Superscript Vilo
•Reformulated pre-Amp
•Small volumes & no
dilution enable use of
entire lysate sample in
subsequent RT/PreAmp
rxns
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Single Cell-CT Kit (400 Reactions)
4 12/23/2011 | Life Technologies™ Proprietary and confidential
•500 L Single Cell Lysis Solution (store at 4ºC)
•50 L Single Cell Stop Solution (store at -20ºC)
•50 L Single Cell DNase I (store at -20ºC)
•150 L Single Cell VILO™ RT Mix (store at -20ºC)
•75 L Single Cell SuperScript® RT (store at -20ºC)
•265 L Single Cell PreAmp Mix (store at -20ºC)
Kit Components 50 and 400 reaction kits
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Single Cell-CT Kit (400 Reactions)
5 12/23/2011 | Life Technologies™ Proprietary and confidential
Starting Material
Samples can be obtained through
− FACS
− Dilution
− Laser capture microdisection (not tested internally)
− Physical selection (eg bead based)
− Laser Ablation (Cyntellect Leap System; not tested internally)
− Mouth pipetting
Up to 10 cells can be used
Input volume of cell (s) should be less than 1 ul
We have validated 5 cell lines (including hESC) for this kit and >20 for the parent kit
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Single Cell-CT Kit (400 Reactions)
6 12/23/2011 | Life Technologies™ Proprietary and confidential
Product Performance
12
14
16
18
20
22
24
26
28
CT
1 Cell
Equivalents
Single Cells 100 cells
20.4
21.6
13.6
Single Cell Detection Occurs with expected sensitivity (6.6 Cts difference from 100
cells). Technical reproducibility of the 100 cell samples and single cell equivalents
is tight (CV of Cell Equivalents is small). Variability of single cells is due to
biological variability in single cells
N=84 single cells
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Single Cell-CT Kit (400 Reactions)
7 12/23/2011 | Life Technologies™ Proprietary and confidential| Life Technologies Proprietary & Confidential | 12/23/2011
SV25 (Olig2-EGFP), a derivative of BG01• Platform line maintains expression of hESC markers
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Single Cell-CT Kit (400 Reactions)
8 12/23/2011 | Life Technologies™ Proprietary and confidential
Grow ESCs on Geltrex plates in CM
until 80-90% confluent
Change media to SFM for 24 hours.
Culture in NAA media until
confluent, changing daily.
Split cells 1:2 using TrypLE, culture
on Geltrex plates.
Once confluent use collagenase,
create neurospheres 150 – 250 um
in Ultra Low Attachment plates.
When cells start to attach to the
plate, split 1:2 onto Geltrex plates.
Continue culturing on Geltrex
plates, splitting 1:2 every 2-3 days.
Dissociate rosettes into single
cells
Day 0 ESC
Day 1 ESC
Day 2 Differentiating ESC
Day 6 Neurospheres
Day 13 Neurospheres
Day 17 Attached
Neurospheres
Day 20 Rosette formation
Day 22 NSC derived
ESC to NSC Workflow 1575 m
Day 0 Day 3
Day 7 Day 11
Day 17 Day 18
Day 21 Day 21
157 mDay 24 GFP Overlay
| Life Technologies Proprietary & Confidential | 12/23/2011
Learn more about the Single Cell-CT Kit (400 Reactions)
9 12/23/2011 | Life Technologies™ Proprietary and confidential
Tested Single Cell Analysis Workflow
• Look at single cells to more closely define profiles, use cells to cell isolation
in 96-well plates, to qPCR in 384-well plates
0 cells 100 cells
10 genes/cell10 plates/time point
•30 “0” cell samples
•30 “100” cell samples
•900 “1” cell samples
Cells-to-CT™ TaqMan®
PreAmp MMx
Gene
Expression
Master Mix
VILO RT
Superscript®
| Life Technologies Proprietary & Confidential | 12/23/2011
Embryonic Stem Cells
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Single Cell-CT Kit (400 Reactions)
10 12/23/2011 | Life Technologies™ Proprietary and confidential
Single cell analysis or Embryonic Stem Cells
100 cells (average CT): 13.7 + 0.2
1 cell low cluster (36 cells): 19.2 + 1.3
1 cell high cluster (48 cells): 27.4 + 1.0
Average CT (84 cells): 21.4 + 4.2
Single cell equivalents (100 samples):
22.8 + 0.3
• Analyzing gene expression profiles en masse gives an average profile
• Obscures or potentially obliterates any differences in single cells
100 cells1 cellACTB
| Life Technologies Proprietary & Confidential | 12/23/2011
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Single Cell-CT Kit (400 Reactions)
11 12/23/2011 | Life Technologies™ Proprietary and confidential
Single cell analysis or Embryonic Stem Cells• Gene variability - large expression range for one gene; size variations do
not account for this, but cell cycle dependent regulation may
100 cells1 cell
ACTB
100 cells1 cell
OCT4
• Cell-to-cell variability - expression profiles are not the same in every cell
• See small sub-populations (OCT4 low expressers)
“technical” variability
22.8±0.3 (6.2 from 100
cell samples)
• Technical variability (from method of detection) needs to be identified
(low here)
| Life Technologies Proprietary & Confidential | 12/23/2011
Learn more about the
Single Cell-CT Kit (400 Reactions)
12 12/23/2011 | Life Technologies™ Proprietary and confidential
10
20
30
40
CT
10
20
30
40
CT
CT
Day 0
Day 14
Day 24
Variation in expression level in single cells
10
20
30
40
GA
PD
GF
AP
NE
S
PA
X6
PO
U5F
1 T
TU
BB
3
UT
F1
ZF
P42
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Single Cell-CT Kit (400 Reactions)
13 12/23/2011 | Life Technologies™ Proprietary and confidential
Noise - Effects on Normalization
• Noise is an inherent part of a biological system and results in cell-to-cell differences
• Extrinsic noise - variations of the levels of transcription factors, polymerases etc. -
results in cell to cell differences for total fluorescence (or total levels of
transcription)
• Intrinsic noise - variation introduced from the act of transcription itself - results in
differences in the levels of independently expressed fluorescent proteins that are
under identical promoters
• Noise causes differences that calls into question the use of “normalization”
• When analyzing a population noise is averaged out making “normalization” more
appropriate.
Elowitz et al., Science 2002| Life Technologies Proprietary & Confidential | 12/23/2011
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Single Cell-CT Kit (400 Reactions)
14 12/23/2011 | Life Technologies™ Proprietary and confidential
100 Cell Data From Day 0
5
10
15
20
25
30
35
GAPDH NES POU5F1 TUBB3 ZFP42
CT
Thirty 100 cell samples show similar expression levels as demonstrated by small center quantiles (left).
Normalized expression levels of each gene to GAPDH expression levels remove some of the sample to
sample variability as shown by smaller box and whisker (right) and show that the gene “profiles” of each
sample are very similar.
•100 cell samples have similar expression levels which
tighten when normalized
-10
-5
0
5
10
15
20
CT
NES-
GAPDH
POU5F1-
GAPDH
TUBB3-
GAPDH
ZFP42-
GAPDH
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Single Cell-CT Kit (400 Reactions)
15 12/23/2011 | Life Technologies™ Proprietary and confidential
Single Cell Data From Day 0
•Single cell samples give wide range of expression levels which spreads
out further when normalized
900 single cell samples show a wide range of expression levels shown by the large box and
whiskers (left). After normalization (right), box and whisker sizes increase as does the
number of outliers
15
20
25
30
35
40
45
GAPDH NES POU5F1 TUBB3 ZFP42
CT
-10
-5
0
5
10
15
20
NES-
GAPDH
POU5F1-
GAPDH
TUBB3-
GAPDH
ZFP42-
GAPDH
CT
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Single Cell-CT Kit (400 Reactions)
16 12/23/2011 | Life Technologies™ Proprietary and confidential
Conclusions and Impacts• There are significant differences from cell-to-cell
• Analyzing gene expression en masse gives an average profile
and masks differences and variability (gene-to-gene and cell-to-
cell)
• Small populations are lost when large populations are averaged
• The TaqMan Single Cell-to-Ct kit:
| Life Technologies Proprietary & Confidential | 12/23/2011
• Optimized reagents provide a simplified workflow for expression
analysis of single cells by qRT-PCR
• Enables transfer of entire cell into each step
• No sample is lost during the reaction which occurs in a single
tube
• Enables the acquisition of statistically significant data sets
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Single Cell-CT Kit (400 Reactions)
17 12/23/2011 | Life Technologies™ Proprietary and confidential| Life Technologies Proprietary & Confidential | 12/23/2011
Normalization Conclusions
•When analyzed en masse, variation in expression level is
reduced when results are normalized to reference genes.
•Expression levels of each gene vary independently within
a single cell
•In single cells normalization increases the variation in
calculated expression level.
•These normalized values are not the same within each
cell and vary depending on the genes compared.
•These results suggest that normalizing single cell data is
not an accurate method of analysis.
Learn more about the
Single Cell-CT Kit (400 Reactions)
18 12/23/2011 | Life Technologies™ Proprietary and confidential| Life Technologies Proprietary & Confidential | 12/23/2011
Acknowledgements
Ron Abruzzese
Richard Fekete
Laura Chapman
Dan Kephart
Andrew Lemire
Penn Whitley
Elena Grigorenko
Ying Liu
Chad MacArthur
Gothami Padmabandu
Jon Chesnut
Mahendra Rao
Janice Au-Young
David Keys
Jonathan Wang
Life Technologies:
2130 Woodward St.,
Austin, TX
12 Gill St. Suite 4000
Woburn, MA
25791 Van Allen Way,
Carlsbad, CA
850 Lincoln Center Dr.,
Foster City, CA
19 12/23/2011 | Life Technologies™ Proprietary and confidential| Life Technologies Proprietary & Confidential | 12/23/2011
Life Technologies, Applied Biosystems and Ambion products are for Research Use Only. Not for
use in diagnostic procedures.
The trademarks mentioned herein are the property of Life Technologies Corporation or their
respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc.
AmpliGrid is a trademark of Beckman Coulter Inc.
NOTICE TO PURCHASER: Limited Use Label License
The products shown in this presentation may be covered by one or more Limited Use Label
License(s). Please refer to the respective product documentation or the Applied Biosystems
website under www.appliedbiosystems.com for the comprehensive license information. By use of
these products, the purchaser accepts the terms and conditions of all applicable Limited Use
Label Licenses. These products are sold for research use only, and are not intended for human or
animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable
product documentation or the respective Limited Use Label License(s).
© 2010 Life Technologies Corporation. All rights reserved.
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Single Cell-CT Kit (400 Reactions)