Single cell analysis has the potential to transform life science research. The standard practice of averaging gene expression resµl ts from many cells can mask subtle, important changes occurring within single cells. To further understand the complexity of biological systems, single cell analysis must be performed. The Ambion® Single Cell-to-CT™ Kit contains a complete validated workflow for gene expression analysis for samples containing between 1-10 cells. Each kit contains reagents for sample preparation, reverse transcription, pre-amplification and qPCR that have been optimized together in a simple workflow that can be completed in only 5 steps.

1. Analyzing Expression Profiles from
Single Stem Cells Using the Single Cell-
to-Ct™ kit
Ron Abruzzese, Ph.D.
Life Technologies
Austin, Texas
1 12/23/2011 | Life Technologies™ Proprietary and confidential

2. Learn more about the
Why Study of Single Cells? Single Cell-CT Kit (400 Reactions)
•Data from ensemble averaging in cell
 Cells are not homogenous measurements can be misleading
• Interesting Biological Research Applications
• Circulating metastatic cells
Research into rare cell
• Fetal cell in maternal blood
or event
• Event within a library
Research using • Archival tissue (FFPE)
scarce, precious • Clinical sample (fresh tumor)
sample • Biomarker discovery
2 | Life Technologies | 12/23/2011 | Life Technologies™ Proprietary and confidential

3. Learn more about the
Single Cell-CT Kit (400 Reactions)
Develop a complete workflow to obtain
statistically relevant single cell data
Benefits
•Optimized reagents
•Superscript Vilo
•Reformulated pre-Amp
Dynabeads®
CD3/CD28 •Small volumes & no
Naive or
dilution enable use of
resting
T cell
entire lysate sample in
subsequent RT/PreAmp
rxns
3 12/23/2011 | Life Technologies™ Proprietary and confidential

4. Learn more about the
Single Cell-CT Kit (400 Reactions)
Kit Components 50 and 400 reaction kits
•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)
4 12/23/2011 | Life Technologies™ Proprietary and confidential

5. Learn more about the
Single Cell-CT Kit (400 Reactions)
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
5 12/23/2011 | Life Technologies™ Proprietary and confidential

6. Learn more about the
Single Cell-CT Kit (400 Reactions)
Product Performance
28
26
24
22
20.4
CT
20
18 21.6
16
13.6
14
12 N=84 single cells
1 Cell Single Cells 100 cells
Equivalents
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
6 12/23/2011 | Life Technologies™ Proprietary and confidential

7. Learn more about the
Single Cell-CT Kit (400 Reactions)
SV25 (Olig2-EGFP), a derivative of BG01
• Platform line maintains expression of hESC markers
7 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

8. Learn more about the Single Cell-CT Kit (400 Reactions)
ESC to NSC Workflow 1575 m
Grow ESCs on Geltrex plates in CM
until 80-90% confluent
Day 0 ESC
Change media to SFM for 24 hours. Day 0 Day 3
Day 1 ESC
Culture in NAA media until
confluent, changing daily.
Day 2 Differentiating ESC
Split cells 1:2 using TrypLE, culture
on Geltrex plates. Day 7 Day 11
Day 6 Neurospheres
Once confluent use collagenase,
create neurospheres 150 – 250 um
in Ultra Low Attachment plates.
Day 13 Neurospheres Day 17 Day 18
When cells start to attach to the
plate, split 1:2 onto Geltrex plates.
Day 17 Attached
Neurospheres Continue culturing on Geltrex
plates, splitting 1:2 every 2-3 days.
Day 20 Rosette formation Day 21 Day 21
Day 24 GFP Overlay 157 m
Day 22 NSC derived
Dissociate rosettes into single
cells
8 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

9. Learn more about the
Single Cell-CT Kit (400 Reactions)
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
Cells-to-CT™ VILO RT TaqMan® Gene
0 cells 100 cells
Superscript® PreAmp MMx Expression
Master Mix
Embryonic Stem Cells
10 plates/time point
•30 “0” cell samples 10 genes/cell
•30 “100” cell samples
•900 “1” cell samples
9 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

10. Learn more about the
Single Cell-CT Kit (400 Reactions)
Single cell analysis or Embryonic Stem Cells
• Analyzing gene expression profiles en masse gives an average profile
• Obscures or potentially obliterates any differences in single cells
ACTB 1 cell
100 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
10 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

11. Learn more about the
Single Cell-CT Kit (400 Reactions)
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
• Cell-to-cell variability - expression profiles are not the same in every cell
• See small sub-populations (OCT4 low expressers)
• Technical variability (from method of detection) needs to be identified
(low here)
ACTB OCT4 “technical” variability
1 cell 1 cell
100 cells 100 cells 22.8±0.3
(6.2 from 100
cell samples)
11 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

12. Learn more about the
Single Cell-CT Kit (400 Reactions)
Variation in expression level in single cells
40
30
CT
Day 0
20
10
40
30
Day 14
CT
20
10
40
30
CT
Day 24
20
10
UTF1
ZFP42
POU5F1
T
TUBB3
NES
GFAP
PAX6
GAPD
12 12/23/2011 | Life Technologies™ Proprietary and confidential

13. Learn more about the
Single Cell-CT Kit (400 Reactions)
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.
13 | Life Technologies Proprietary & Confidential | 12/23/2011 Elowitz et al., Science 2002Technologies™ Proprietary and confidential
12/23/2011 | Life

14. Learn more about the
Single Cell-CT Kit (400 Reactions)
100 Cell Data From Day 0
•100 cell samples have similar expression levels which
tighten when normalized
35 20
30 15
25 10
CT
CT
20 5
15 0
10 -5
5 -10
GAPDH NES POU5F1 TUBB3 ZFP42 NES- POU5F1- TUBB3- ZFP42-
GAPDH GAPDH GAPDH GAPDH
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.
14 12/23/2011 | Life Technologies™ Proprietary and confidential

15. Learn more about the
Single Cell-CT Kit (400 Reactions)
Single Cell Data From Day 0
•Single cell samples give wide range of expression levels which spreads
out further when normalized
45 20
40 15
35 10
CT
CT
30 5
25 0
20 -5
15 -10
GAPDH NES POU5F1 TUBB3 ZFP42 NES- POU5F1- TUBB3- ZFP42-
GAPDH GAPDH GAPDH GAPDH
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 12/23/2011 | Life Technologies™ Proprietary and confidential

16. Learn more about the
Single Cell-CT Kit (400 Reactions)
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:
• 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
16 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

17. Learn more about the
Single Cell-CT Kit (400 Reactions)
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.
17 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

18. Acknowledgements
Life Technologies:
Ron Abruzzese 2130 Woodward St.,
Richard Fekete Austin, TX
Laura Chapman
Dan Kephart
Andrew Lemire
Penn Whitley
Elena Grigorenko 12 Gill St. Suite 4000
Woburn, MA
Ying Liu 25791 Van Allen Way,
Chad MacArthur Carlsbad, CA
Gothami Padmabandu
Jon Chesnut
Mahendra Rao
Janice Au-Young 850 Lincoln Center Dr.,
David Keys Foster City, CA
Jonathan Wang
18 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

19. Learn more about the
Single Cell-CT Kit (400 Reactions)
Legal Statements
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.
19 | Life Technologies Proprietary & Confidential | 12/23/2011 12/23/2011 | Life Technologies™ Proprietary and confidential

20. Learn more about the
Single Cell-CT Kit (400 Reactions)
www.lifetechnologies.com
20 12/23/2011 | Life Technologies™ Proprietary and confidential