PCR-Select™ cDNA Subtraction-Generated Libraries Enable Detection of
Physiologically Relevant Gene Transcripts in an In Vitro Model of Angiogenesis
C. N. Hahn1, Z. J. Su1, C. J. Drogemuller1,
A. Tsykin1,3, S. R. Waterman1, P. J. Brautigan1,
S. Yu1, G. Kremmidiotis2, A. Gardner2,
P. J. Solomon3, G. J. Goodall1,4, M. A. Vadas1,4
and J. R. Gamble1,4
1 Human Immunology, Hanson Institute, Frome Rd,
Adelaide, SA, 5000 Australia.
2 Bionomics Limited, 31 Dalgleish St, Thebarton,
SA, 5031 Australia.
3 School of Mathematics, The University of Adelaide,
Adelaide, SA, 5005 Australia.
4 Department of Medicine, The University of Adelaide,
Adelaide, SA, 5005 Australia.
In this study, we have used SMART™ cDNA
synthesis technology to amplify small amounts
of total RNA, and the PCR-Select cDNA Subtraction Kit to generate enriched and normalized
cDNA libraries in conjunction with microarray
analysis to facilitate selection of genes regulated
during in vitro angiogenesis. This strategy allowed
the construction of custom-made microarrays
enriched for physiologically relevant targets.
Virtual Northern (VN) blots derived from
SMART cDNA were then used to measure
the magnitude of differential gene expression.
Microarray analysis of gene expression is a
powerful tool for studying global changes
in cellular mRNA levels during developmental processes or following endogenous
or exogenous stimuli. However, most
microarrays, even those that represent
the majority of known and predicted
protein-coding genes, do not account for
alternative splicing, promoters, and polyadenylation termination, which may vary
during development, between cell types
or following a stimulus. Approaches that
enable identification of regulated novel
as well as known or predicted transcripts
that are relevant to the cell type or process
being studied include differential display,
serial analysis of gene expression (SAGE),
and suppression subtractive hybridization
(SSH). Each of these procedures requires
considerable amounts of starting material
(e.g., 5–50 µg total RNA).
Generation of custom-made
angiogenic microarrays using
PCR-Select cDNA-subtracted
libraries
The strategy for designing custom-made
microarrays and identifying differentially
expressed genes during the capillary tube
formation process is outlined in Figure
1 (1). Human umbilical vein endothelial cells (HUVEC) were plated onto a
3D-collagen matrix to induce capillary
tube formation. RNA was then isolated at
times 0, 0.5, 3, 6, and 24 hr. Clontech’s
SMART PCR cDNA Synthesis Kit
(Cat. No. 634902) was used to make
sufficient cDNA from 1 µg of total RNA.
A single amplification, as used here, does
not significantly reduce the complexity of
the cDNA population (2). The Clontech
PCR-Select cDNA Subtraction Kit
(Cat. No. 637401) was then used to
perform suppression subtractive hybridization between adjacent time points in
both the forward and reverse orientations.
Four forward and four reverse libraries
were then generated by digesting the
subtracted cDNAs. Individual clones
(10,000) were picked from the four forwardsubtracted libraries, the inserts amplified
by PCR, and the products spotted onto
glass slides. These slides were probed with
RNA isolated from cells taken at 0, 0.5, 3,
6, and 24 hr, and labeled with Cy3/Cy5
dyes. Labeled RNA from each time point
was compared to RNA from time 0.
MMP10
A
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hr
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polyA 3'
5'
5'
5
10
15
20
25
Modified oligo(dT)
polyA
Template switching
polyA
GGG
CCC
PCR
Suppression Subtractive Hybridization
(PCR-Select™ cDNA Subtraction Kit)
Subtracted Libraries
(forward & reverse)
Isolate & Grow Individual Clones
PCR Amplify Insert cDNA
Microarray
(spot insert cDNA onto slides)
Probe using Cy3/Cy5-Labeled RNA
Scan & Analyze Data
(generate expression profiles)
Sequence Regulated Clones to Identify
Quantify Gene Expression Profiles
(VN Analysis and Q-RT-PCR)
Figure 1. cDNA synthesis and amplification
strategy using the SMART PCR cDNA
Synthesis Kit. A schematic flowchart
describing the identification of differentially expressed genes during in vitro
angiogenesis via custom-made microarrays
using SMART cDNA synthesis technology
and the PCR-Select cDNA Subtraction Kit.
5
10
15
EGR1
n = 35
20
n = 37
3
2
1
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–2
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RT
SMART II
5'
GGG
CCC
2
2
0
5'
PTGS2
n = 28
8
0
5
10
15
20
25
10
15
20
25
Figure 2. Comparison of angiogenesis time course profiles from microarray, quantitative
RT-PCR (Q-RT-PCR), and Virtual Northern (VN) analysis. Expression profiles for MMP10,
PTGS2, and EGR1 from microarrays (Panel A) were quantified using Virtual Northern (VN)
analysis (Panel B) and Q-RT-PCR (Panel C). Plotted is fold induction (log2) with respect
to time 0 (y-axis) vs. time (hr: x-axis). Panel A. Profiles from cDNA fragments relating
to the same gene were overlaid. The total number of profiles plotted (n) of individual
clones relating to each gene is shown in the top right corner. Panel B. VN blots were quantified by phosphor imaging, standardized to a time-matched peptidylprolyl isomerase A
(Cyclophilin A) control, and the results plotted above each blot. Panel C. Q-RT-PCR
was performed in triplicate and the average standardized to a PPIA control.
Clontech Laboratories, Inc. • www.clontech.com
CR692130
10
Poly A+ mRNA
5'
GGG
Reprinted from Clontechniques April 2006 PCR-Select™ cDNA Subtraction-Generated Libraries Enable Detection of
Physiologically Relevant Gene Transcripts…continued
PCR-Select cDNA-subtracted
microarrays enable identification of relevant known
and novel genes in in vitro
angiogenesis
VN
8
6
4
2
0
–2
–4
Following background subtraction and
normalization of data, expression profiles
during the 24 hr capillary tube formation were plotted for all 10,000 clones.
1,728 clones were chosen for sequence
identification and these represented over
500 genes. Many genes were represented
multiple times and displayed the same
expression profile, although the magnitude of expression varied considerably.
Profiles for highly regulated MMP10,
PTGS2 and EGR1 genes, are shown
(Figure 2A). VN blots derived from
SMART amplified cDNAs were used
to confirm and accurately quantify
expression profiles (Figure 2B). Profiles
were also quantified using quantitative
RT-PCR (Q-RT-PCR) (Figure 2C).
A comprehensive comparison of quantification of mRNA via VN analysis and
Q-RT-PCR demonstrated a very good
correlation across several orders of magnitude (Figure 3). The advantages
TIEG
A
1.5
0.5
–0.5
B
hr
0
0
5
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0.5
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20 25
24
0.5
hr
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ARHGAP24
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–1.0
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D
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2.5
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sFLT1
1.0
0
–0.5
2
Q-RT-PCR
ESM1
3.0
–0.5
0.5
0
This approach has allowed the identification of known differentially expressed
genes in angiogenesis (e.g., PTGS2,
PTRF
1.5
–2
Notice to Purchaser
of VN analysis in this approach include
no need for prior knowledge of the cDNA
sequence or for synthesis of primers
for quantification, as well as excellent
sensitivity and the ability to reprobe blots
multiple times (up to 8 times in our hands).
0.5
2.5
–4
2.0
JAG1
C
Slope = 1.05 ± 0.06
R2 = 0.78
Figure 3. Virtual Northern (VN) analysis
closely correlates with quantitative-RT-PCR
(Q-RT-PCR). VN analysis and Q-RT-PCR
were used to measure the levels of gene
expression for 75 samples (representing
13 different genes) in the capillary tube
formation assay. These were standardized
to a PPIA control and expressed as fold
induction with respect to time 0. A scatter
plot is shown, plotting fold induction (log2)
as determined by VN blot vs. Q-RT-PCR.
Linear regression was used to determine
the slope of the line of best fit.
CMG2
2.0
1.5
1.0
0.5
0
–0.5
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of Clontech Laboratories, Inc. Clontech is a Takara Bio
Company. Copyright 2006.
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Figure 4. Virtual Northern (VN) blots accurately and reproducibly confirm microarray
profiles. The expression time courses for genes differentially regulated during angiogenesis were determined using microarray (Panels A and C) and VN analysis (Panels B and D).
In Panels A and C, the fold induction (log2) is plotted against time (hr) following stimulation of angiogenesis. In Panels B and D, VN blots and time points (hr) are shown. Despite
various banding patterns or smears, the regulation profiles closely mirrors those obtained
using microarray analysis.
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For all licensing information, visit www.clontech.
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EGR1, CMG2, ESM1, JAG1, and FLT1)
(Figure 4) as well as novel genes (e.g.,
ARHGAP24), previously unrecognized
in capillary tube formation (1), but now
shown to be a critical player (3). For a
number of genes, including soluble FLT1
(sVEGFR1), alternative downstream
polyA+ sites were identified, while for
others alternative splicing and extended
5’UTRs were found (not shown). Genes
represented by low-abundance transcripts
were also detected, probably as a result
of the subtraction normalization process.
Limited amounts of starting RNA
were then successfully amplified using
SMART-amplified cDNA synthesis to
facilitate generation of subtracted libraries
via PCR-Select cDNA subtraction for
construction of angiogenic custom-made
microarrays. Probing these arrays enabled
confirmation of known angiogenic players
and the identification of novel genes and
alternative gene transcripts together with
their expression profiles. In some situations, such as this one, VN analysis is an
attractive alternative to Q-RT-PCR for
accurate quantification of microarray data,
and is definitely superior in sensitivity
to Northern blot analysis.
References
1. Hahn, C. N., et al. (2005) Physiol. Genomics
22(1):57–69.
2. Puskas, L. G., et al. (2002) BioTechniques
32(6):1330–1340.
3. Su, Z-J., et al. (2004) Proc. Natl. Acad. Sci. (USA)
101(33):12212–12217.
Reprinted from Clontechniques April 2006