TaqMan®-based miRNA profiles classify
mouse ES and differentiated cells
Caifu Chen, Dana A. Ridzon, Yu Liang, Linda Wong, Karl J. Guegler, Advanced Research & Technology, Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, USA;
William M. Strauss, Department of Molecular, Cellular, & Developmental Biology, University of Colorado, Boulder, CO 80309, USA
ABSTRACT
Figure 1. TaqMan®-based miRNA profiles classify ES cells,
differentiated embryoid bodies (EB) and tissues (embryo, brain etc.)
1
expression (≥ 2 fold) only in 3 miRNAs after ES cell differentiation into EBs.
Tissues
ES
EB
85
Tissues
ES
EB
169
Tissues
ES
EB
Highly expressed
Intermediately expressed
Figure 2. Mouse ES cell-, EB- or tissue-specific MicroRNAs
EB
ES-specific
EB
Tissues
ES
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15
Tissues
ES
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15
miR-24
miR-23bN
miR-26a
INTRODUCTION
miR-290
MicroRNAs are endogenous RNAs of ~22 nucleotides that
play important regulatory roles in animals & plants (1). We
propose that miRNAs are likely important regulators for stem
cell self-renewal. Our hypothesis is based on the observation
that distinct sets of miRNAs are specifically expressed in
pluripotent ES cells but not in differentiated EBs (2), and that
EBs express miRNAs not found in ES cells. To test this
hypothesis we have conducted genome-wide expression of
252 miRNAs in mouse ES cells, EBs, and adult tissues. This
survey of miRNA expression utilized TaqMan miRNA assays
to profile miRNA expression signatures or fingerprints.
miRNA expression signatures in single cells will be useful in
determining tissue/cell identity and monitoring the ES
differentiation.
miR-302b
miR-30a-5p
miR-302d
miR-30c
miRNA genes: 254 mouse and/or human miRNAs.
RNA samples: Six from mouse tissues, 13 from mouse ES
lines, and 5 from embryoid bodies at day 6 or 9.
Stem cell differentiation: Aggregated ES cells were
cultured under feeder- and serum-free condition (Kanno et al.
2004). Adherent (Ad) and non-adherent (NAd) cells were
harvested at 6 and 9 days.
RT-PCR: The assay includes two steps, RT and PCR (Figure
1). RT reactions were incubated for 30 min each, at 16oC and
at 42oC. Real-time PCR was performed on an AB 7900HT
Sequence Detection System.
Data analysis: Total RNA input was normalized based on CT
values of 18S rRNA assay. All CT values with 35 or higher
are adjusted to 35. The fold-change was calculated based on
CT changes of mean medium CT minus individual CT of a
miRNA. Agglomerative hierarchical clustering was performed
using CLUSTER program (3).
Less or unexpressed
Total RNA input was normalized based on the CT values of 18S rRNA. The fold-change was
calculated based on CT changes of mean medium CT minus individual CT.
miR-302c
MATERIALS AND METHODS
Figure 3. Highly conserved miRNA expression profile in 13 mouse
ES cell lines. Most miRNAs in ES cells are expressed at a low level.
However, ¼ (61) miRNAs are highly expressed in ES. MicroRNA
expression signature, together with ES-specific mRNA marker
Oct3/4 confirmed the stemness of these ES lines. However, the
variability of expression levels in some miRNAs, not mRNA genes
suggests that miRNAs could be a better indicator to monitor
spontaneous ES differentiation during cell culture.
miR-26aN
miR-367
miR-26b
miR-30bN
miR-302a
miR-191
miR-295
miR-30b
miR-293
miR-106b
miR-294
miR-15a
miR-182*
miR-126
miR-133a
Tissue-specific
miR-223
miR-153
Figure 4. MicroRNA expression changes of five ES cell lines upon
differentiation
miR-139
miR-341
miR-98
miR-154
miR-187
miR-129
miR-137
miR-27aN
miR-34cN
Liver
miR-208
miR-378
miR-197
EBs:
1. EB35d9_Ad
2. EB60d9_NAd
3. EB60d6_Ad
4. EB60d6_NAd
5. EB35d6_Ad
ES lines
6. ES60b
7. ES60a
8. ES35
9. ES60c
Tissues:
10. Liver
11. Heart
12. Lung
13. Kidney
14. Brain
15. Embryo
miR-105
Highly expressed
Intermediately expressed
Expression
miRNA
Down
miR-9*
miR-9
Less or unexpressed
Table 1. MiRNA expression in mouse ES and differentiated cells
Mouse tissues
Expressed
Up
#101
#316
#317
EB
EB
EB
#318
#331
EB
EB
ES
Day3
Day6
Day9
ES
Day3
Day6
Day9
ES
Day3
Day6
Day9
ES
Day3
Day6
Day9
ES
Day3
Day6
Day9
TaqMan® miRNA assays have been developed using stemloop primers for reverse transcription (RT) followed by realtime PCR. A total of 254 mouse and human miRNA assays
were examined with thirteen different mouse embryonic stem
cell (ES) lines and their differentiated embryoid bodies (EBs)
and six mouse tissues. MicroRNA expression profiles can
classify the ES cells, differentiated EBs and adult tissues.
We have identified a number of ES-specific and
differentiation-related miRNA candidates that could be used
as molecular markers to determine ES cell identity and to
monitor the differentiation. There exists a highly conserved
miRNA expression signature in 13 mouse ES lines. This
signature, when compared to all cellular lineages, includes
three components of miRNA expression: an unchanging pandevelopmental microRNA expression pattern, increasingly
complex somatically acquired microRNA expression pattern,
and stem cell specific microRNA expression pattern. Only ¼
miRNA genes are highly expressed in ES cells, and during
development an increasingly elaborate miRNA signature is
expressed. The stem cell specific expression of a small set
of microRNAs is lost in an apparently coordinate fashion
during development and does not reappear in any somatic
lineage. Based on the elucidation of this regulated miRNA
molecular signature, it seems likely that there is a significant
role for miRNA action in the early embryo.
RESULTS
miR-152
miR-152N
miR-210
REFERENCES
miRNAs*
ES
EB
Brain Embryo Heart Kidney Liver
Lung
No.
119
149
206
178
175
189
147
201
%
47
59
82
71
69
75
58
80
* miRNAs are defined as “Expressed” if their CT values are less than 35 (an estimate
of >1-5 copies per cell). A total of 252 miRNAs are used to calculate % of expressed
ones in a tissue or cell type
1. Bartel, D. 2004. Cell 116: 281-297
2. Houbaviy et al. 2003. Developmental Cell 5:351
3. Eisen et al. 1998. PNAS 95:14863-14868
NOTES
For Research Use Only. Not for use in diagnostic procedures. The PCR process and 5'
nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F.
Hoffmann-La Roche Ltd. Applied Biosystems, AB and FAM are trademarks of Applera
Corporation or its subsidiaries in the US and/or certain other countries. TaqMan is a
trademark of Roche Molecular Systems, Inc. © 2005 Applied Biosystems. All rights reserved.
127PR08-02