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From the Department of Medical Biochemistry,* Göteborg University; and AngiogeneticsAB, Göteborg, Sweden
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Abstract |
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 Top Abstract Materials and MethodsResults and DiscussionReferences |
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In this article we present a procedure for microarray hybridization of RNA extracted from cells isolated with laser microbeam microdissection (LMM) and laser pressure catapulting (LPC).5 This procedure offers several important advantages compared to previously used microdissection methods:6-8 the sample is not in contact with any part of the equipment or the collector device during the isolation process thus dramatically reducing the risk of contamination. LMM also allows cells of any shape and size (down to pieces of chromosomes) to be cut out and catapulted. Laser microdissection offers precise control as the cells are picked individually from histological sections. No cell-specific markers are needed for the cell isolation, even populations that are recognized by morphology alone can be isolated purely. Contrary to fluorescence-activated cell sorting or magnetic bead sorting, the tissues are not exposed to collagenase digestion before cell isolation, but are fixed or frozen in their native environment conserving the RNA profile in a true in vivo state. We are primarily interested in cell fate decisions and differentiation that typically occurs in clusters of cells located in slender embryonic structures.9 As these structures are not preserved without fixation the procedure was optimized for fixed material, which in turn makes it applicable to most cell populations. As a proof of principle, expression profiles from cells in the mouse embryonic dorsal aorta at the onset of vascular smooth muscle cell (VSMC) induction was compared to the expression profile from mesenchymal cells located one cell diameter further away from the aorta lumen. Genes encoding endothelial markers, smooth muscle cell markers, and basement membrane proteins, were consistently overexpressed in the aorta cells, confirming the accuracy of the profiles. No such markers were overexpressed in the mesenchymal cells.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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C57BL/6 mice were housed at the Department of Experimental Biomedicine at Göteborg University according to Swedish animal research regulations. All experiments have been approved by the Swedish Research Animal Ethical Committee (Drnr: 213-2000). The morning of vaginal plug detection was counted as E0.5.
Fixation
The following fixatives were tested: zinc-fix (5 g ZnCl2, 6 g ZnAc2 x 2H2O, 0.1 g CaAc2, in 1 L of 0.1 mol/L Tris, pH 7.4), methanol, 70% ethanol, acetone, 4% paraformaldehyde, Formoys (60 ml EtOH, 10 ml HAc, 30 ml of 40% formaldehyde), Carnoys (50 ml EtOH, 25 ml HAc), and methacarn (60 ml EtOH, 30 ml chloroform, 10 ml HAc). Animals were dissected in ice-cold phosphate-buffered saline. Tissues were immersed in respective fixative and left overnight at 4°C.
RNA Recovery and Quality Measurements
Total RNA was extracted from P14 mouse kidneys and hearts with the Qiagen RNeasy mini kit (VWR International AB, Stockholm, Sweden). RNA content was quantified with UV-spectrophotometric analysis (A260), and recovery rates are presented as percentage of RNA content in directly homogenized tissue. RNA integrity was analyzed with electrophoresis using the NorthernMax kit (Ambion Ltd, Cambridgeshire, UK). Five µg of total RNA was loaded on 1% agarose gels. The RNA quality was evaluated by incorporation of 32P-labeled CTP in the first and second strand cDNA-synthesis reaction. cDNA was generated with the replacement method according to standard protocols using a polydT primer. 32P-labeled CTP was added to a final concentration of 1 µCi/µl of 10 mmol/L dNTP mix. The 32P-labeled cDNA was size-fractionated on a 0.8% agarose gel, transferred to nylon filter, and analyzed with a phosphoimager according to standard procedures.
Small amounts (<1 µg) of total RNA were extracted with the Micro RNA Isolation Kit (Zymo Research, Orange, CA). RNA quantities were measured with Ribo-Green RNA Quantitation Kit (Molecular Probes Europe BV, Leiden, The Netherlands) in a fluorometer (TD-360; Turner Designs Inc, Sunnyvale, CA). All procedures were performed according to the manufacturers’ instructions.
LMM and LPC
E9.5 mouse embryos (C57BL/6) were zinc-fixed (4°C) overnight,
and then incubated 4 hours in zinc-fix with 30% sucrose. Embryos were
mounted in Tissue-Tek OCT compound (Sakura Finetek, Torrence, CA) and
stored at -80°C until sectioning. Frozen sections (10 µm) were
mounted on plus-charged slides (SuperFrost plus; Menzel-Gläser,
Braunschweig, Germany), and left to dry for 30 minutes in room
temperature before storage at -80°C in boxes with silica gel. Every
sixth section was mounted on a reference glass that was stained for
-smooth muscle actin (-SMA). For laser capturing, the slides were
put into zinc-fix on ice for 5 minutes to dissolve the OCT that
otherwise interferes with LMM. Next, the slides were dehydrated for 30
seconds in 70%, 95%, and 99.5% ice-cold ethanol, respectively,
incubated 1 minute in xylene, and dried at room temperature. Five
sections were mounted on each slide and were captured in one session
using the PALM Robot-MicroBeam system (P.A.L.M. Mikrolaser Technology
AG, Bernried, Germany). The LPC-collected cells (
200) were solved in
40 µl of lysis buffer (Micro RNA isolation kit; Zymo) and stored at
-80°C. The process was repeated until 1000 cells were collected.
RNA Extraction and T7 RNA Amplification
LMM-isolated cells in lysis buffer were thawed and centrifuged briefly before the RNA was extracted using the Micro RNA isolation kit (Zymo) according to the manufacturer’s protocol. T7 RNA (aRNA) amplification was performed in three cycles mainly according to the protocol given in Wang and colleagues10 but with the following modifications: 0.5 µl (5 µg/µl) of linear acrylamide (Ambion Ltd.) was added in the first step of oligo-dT(15)-T7 primer annealing. After second-strand synthesis, double-stranded cDNA was phenol-chloroform-isoamylalcohol extracted once and washed three times with RNase-free water (Ambion Ltd.) on Microcon 100 columns (Millipore AB, Sundbyberg, Sweden). The final volume was adjusted to 16 µl. After in vitro transcription with the T7 Megascript Kit (Ambion Ltd.) for 4 hours at 37°C, the reaction mixture was mixed with 460 µl of lysis buffer (GeneElute kit; Sigma-Aldrich Chemie GmbH, Munich, Germany) and the aRNA was purified according to the RNA isolation protocol provided by the manufacturer. aRNA was eluted from the column with 50 µl of water and vacuum-dried in the presence of 60 U of RNasin (Promega UK, Southampton, UK) to a volume of 5 µl. Subsequent rounds of amplification were performed as described elsewhere.10 However, cDNA purification, in vitro transcription, and aRNA purification were performed as for the first round. A detailed protocol can be downloaded from http://cbz.medkem.gu.se/lindahl/protocols.
Target Labeling and Microarray Hybridization
Five µg of aRNA or 100 µg of total RNA was primed with 5 µg of random hexamer (Promega UK) or 2 µg of oligo-dT primer, respectively, and labeled in a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech AB, Uppsala, Sweden) in a volume of 30 µl, according to standard protocols (http://cmgm.stanford.edu/pbrown). The differently labeled targets were combined, mixed with 1 µl of 10 µg/µl yeast tRNA, 1 µl of 10 µg/µl polyA RNA, vacuum-dried, and resuspended in 20 µl of DIGeasy hybridization buffer (Roche Diagnostics GmbH, Mannheim, Germany). The hybridization mix was placed at 100°C for 2 minutes and then at 37°C for 30 minutes before being added to the chip. Before hybridization, the glasses were rehydrated by placing them array-side down for 15 minutes over 1x standard saline citrate (SSC), fast-dried by placing them array-side up for 10 seconds on a 100°C heat block, and then baked at 80°C for 4 hours and UV cross-linked (300 mJ).
Hybridization was performed in a 40°C water bath for 12 to 18 hours under lifter coverslip (Histolab, Göteborg, Sweden) in ArrayIT hybridization cassettes (TeleChem International Inc, Sunnyvale, CA). After hybridization, the slides were washed in 2x SSC, 0.1% sodium dodecyl sulfate for 5 minutes at room temperature, 1x SSC for 5 minutes, and finally in 0.1x SSC for 5 minutes.
Array Scanning and Data Presentation
The slides were scanned (ScanArray 3000; Packard Bioscience, Meriden, CT) at laser intensity and photomultiplier tube voltage settings giving the best dynamic range for each chip in respective channel. Image segmentation and spot quantification was performed with the ImaGene software (Biodiscovery, Marina Del Rey, CA). After median local background subtraction, the log2-transformed ratios (Cy3intensity/Cy5intensity) were plotted versus the mean log2 intensities 0.5*(log2Cy3intensity + log2Cy5intensity). The ratios were normalized for signal intensity variation in a non-linear intensity-dependent way using the loess function in the S-Plus software (MathSoft Inc, Surrey, UK) as described elsewhere (Dudoit S, Yang YH, Callow MJ, Speed TP, technical report no. 578, August 2000, Department of Biochemistry, Stanford University School of Medicine). A ratio versus intensity plot amounts to a 45° rotation of a log2Cy3intensityversus log2Cy5intensity plot, followed by scaling of the coordinates. This representation allows for intensity-dependent nonlinear normalization, but it also directly visualizes the actual expression ratios throughout the entire 16-bit range of the scanned microarray images.
Statistic Analysis
Mean log2-transformed ratios were calculated from repeated independent experiments. The genes were individually evaluated for overexpression by t-tests, which is applicable because the log-transformed ratios are approximately normally distributed.
Evaluation of T7 Amplification
Expression profiles were generated from nonamplified and amplified
samples in the following way: the amplified heart and kidney RNA was
compared in four hybridization experiments using the independently
amplified samples (hybridizations A1 to
A4). Similarly, the nonamplified heart and kidney
total RNA was compared in four independent hybridizations
(hybridizations T1 to T4).
Mean log2-transformed expression ratios were
calculated for both categories (see Figure 3, a, e and f
).
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).
For evaluation of the amplification effect on expression ratios between
two samples (preservation of ratios), a ratio versus ratio plot was
generated and the correlation coefficent was calculated from the mean
log2-transformed expression ratios
T1-4 and A1-4 (Figure 3g)
.
To assess intensity-dependent correlation of the ratios, all individual
arrays were compared, and ratio versus ratio plots for each
pair were generated. Six pairs described reproducibility of
nonamplified ratios (T to T comparisons). Another six pairs described
reproducibility of amplified ratios (A to A comparisons). Finally, 16
plots described the correlation between nonamplified and amplified
ratios (T to A comparisons). Correlations between expression ratios in
the A versus A, T versus T, and T versus A pairs were calculated in an
intensity-dependent way, as follows: Each ratio versus ratio plot was
divided into 10 groups based on order of abundance. The 10% most
abundant clones were assigned to group 1, next 10% to group 2, and so
forth. The T to A plots were grouped according to order of abundance in
the amplified experiments. For each group, the Pearson correlation
coefficient was calculated and plotted against the
log2 intensity distribution. The curves shown in
Figure 3h
are the loess regression lines (S-Plus software; MathSoft
Inc, Surrey, UK) through the correlation coefficient plots for each
category (T to T, A to A, and T to A).
Microarray Chip Design
The chips were printed with 1350 random chosen mouse
sequence-verified expressed sequence tags from the I.M.A.G.E.
consortium (purchased from Invitrogen Ltd, Renfrewshire, Scotland),
2400 nonsequenced clones from a normalized E16.5 head library
(generously provided by Dr. Oliver Renner, Max-Planck Institute for
Physiological and Clinical Research, Bad Nauheim, Germany), 5
yeast genes, and 100 selected control genes. The clones were polymerase
chain reaction (PCR) amplified, purified, and resuspended in 0.2%
sarkosyl, 3x SSC and printed with the GMS 417 spotter (Affimetrix,
Santa Clara, CA) onto 
-amino propyl silane-coated CMT-Gap slides
(Corning International, London, UK).
Quantitative PCR
TaqMan PCR primers and labeled probes (5' 6-FAM, 3' 6-TAMRA) were designed for 11 genes using the Primer Express software and purchased from Applied Biosystems (Applera Sweden, Stockholm, Sweden) (sequences can be found at http://cbz.medkem.gu.se/lindahl/taqman). The mRNA sequences were obtained from the Celera database.
After laser microdissection and three rounds of RNA amplification, 60 ng of aorta and mesenchyme aRNA was used for first-strand cDNA synthesis. The aRNA was mixed with 4 µl of first strand buffer, 4 µl (500 µg/ml) of random hexamers (Promega UK), 2 µl of dithiothreitol, 1 µl of RNasin (Promega UK), 2 µl of ultrapure dNTPs mix (Clontech, Palo Alto, CA), and water to a final volume of 18 µl. The mixture was incubated at 65°C for 10 minutes followed by 42°C for 1 minute. Two µl of Superscript II (Invitrogen Ltd) was added and incubation at 42°C was continued for 1 hour.
The 50-cycle TaqMan PCR assay was performed in 20-µl reactions in a 384-well microtiter plate under conditions recommended by the manufacturer using 10 µl TaqMan 2x PCR Master Mix (Applera Sweden), 6 pmol of each primer, 2 pmol of probe, and 1.5 ng of cDNA (1/40 of the cDNA reaction mix) using the AB1 PRISM 9700HT realtime PCR cycler (Applied Biosystems, Foster City, CA). Each assay was repeated three times and the mean CT-values were used for further calculations. For each amplicon a standard curve was determined using eight serial dilutions in triplicate of a mixed cDNA template obtained from heart, kidney, and brain total RNA. The relative number of target copies in each sample was interpolated from its detection threshold (CT) value using the standard curve. Expression ratios between aorta and mesenchyme target copies were calculated and changes <20-fold were plotted against the fold changes measured on the microarray. The TaqMan measured ratios were normalized with respect to the offset of the linear fit through this plot.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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The main challenge for obtaining expression profiles from
laser-microdissected cells is the limited amount of RNA that can be
extracted, and it is therefore important to eliminate RNA losses at any
stage. It is equally important to maintain the integrity of the
transcripts because reverse transcription is a prerequisite for
amplification and target labeling. Several fixatives were compared to
evaluate total RNA recovery, RNA quality and the RNA performance as a
template for cDNA synthesis (Figure 1
; a
to c). Generally, compounds with good recovery rates also preserved the
integrity of the RNA and generated long cDNA products. Precipitating
agents such as methanol, ethanol, and acetone efficiently recovered RNA
with preserved integrity, which is in line with previous
reports.11
Cross-linking agents such as 4%
paraformaldehyde and Formoys were not as favorable and failed to
produce but minor amounts of RNA, also in agreement with the
literature.11
Surprisingly, a zinc-based fixative
performed best in our test.12
RNA was recovered almost as
efficiently as after direct homogenization, and the synthesized cDNA
was of comparable length. The protection against endogenous RNases was
sufficient for RNA extraction from kidneys and whole embryos, but not
from pancreas, suggesting that zinc-fix has a moderate protective
ability (data not shown). Contrary to the literature,13
methacarn and Carnoys repeatedly produced low amounts of RNA in our
hands (Figure 1
; a to c).
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. Paraffin-embedded
samples also generated fair amounts of cDNA, but not comparable to
frozen samples (data not shown). Although tissue morphology was
superior in paraffin sections, frozen sections were of sufficient
quality (Figure 1d)
. Most importantly, the cells preserved their
relative positions despite microscopic rifts.
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Experimental Procedure and RNA Content
An experimental procedure for LMM and LPC isolation of cells from
zinc-fixed frozen sections was worked out (Figure 2)
. Most of the steps
are obvious necessities such as fixation, OCT embedding,
cryosectioning, LMM/LPC, and RNA extraction. As LPC is most efficient
with dry samples the sections were taken through a dehydration series
before LMM. OCT interferes with laser cutting, and must be removed by 5
minutes of incubation in zinc-fix before dehydration. Total RNA content
was measured at every step in the procedure to identify bottlenecks and
to minimize losses (Figure 2)
. Generally, the RNA was stable when the
cells were dry or frozen. RNA recovery was impaired at three
steps—fixation, LMM/LPC, and RNA extraction. The evaluation of
LMM/LPC and RNA extraction is discussed below. Fixation that reduces
RNA recovery by 20% has already been covered.
Laser microdissection with the PALM Robot-MicroBeam system is performed
in two steps. First, the cells are outlined with a cutting laser in a
process called LMM.14
Second, the cells are vertically
transferred into a collecting device held in place above (no contact)
the specimen in a process called LPC. The collector is coated with
sterile-filtered mineral oil (Sigma-Aldrich Chemie GmbH) to provide a
sticky surface for the catapulted cells. To exclude that degradation
occurs during LPC, consecutive zinc-fixed frozen sections were either
deposited in mineral oil on the collector for 1 hour at room
temperature, or deposited directly into homogenization buffer for 1
hour at -20°C before RNA extraction and quantification. No loss
occurred in the oil (data not shown). The efficiency of LPC was
initially a concern because we failed to localize the catapulted cells.
A novel collector was designed to optimize cell recovery (for details
see http://cbz.medkem.gu.se/lindahl/device). The key
features of the new collector are a flat surface and free edges (no
rim). This reduced the distance from specimen to collector surface from
2 mm to <100 µm. As a result, the captured cells were easy to spot
and count after catapulting. Furthermore, the short distance allowed
LPC with considerably less energy, which reduced the frequency of
contamination caused by unintended catapulting of neighboring cells.
With our specimens, 85% of the cells could be localized on the
collector.
RNA isolation and purification can be achieved through several principally different protocols. We consistently lost 40% or more of the RNA with protocols involving phenol/chloroform extraction, whereas RNA-binding matrices in spin columns typically recovered >80%. We decided to omit DNase treatment of the samples, as we otherwise had to include a phenol/chloroform extraction. We argued that: 1) DNA is a poor template for reverse transcriptase enzymes and that incorporation of T7 promotor-containing primers into DNA-encoded double-stranded cDNA is rare. 2) DNA-derived signals that bind to spots on the array will not introduce any cell-type-specific signatures, and will thus be neutral to the expression profile.
Total RNA content was quantified in mouse E9.5 dorsal aorta cells
that were later captured for gene expression profiling. The numbers in
Figure 2
are estimates based on measurements of RNA and DNA content in
laser-microdissected cells, measurements of the average cell RNA
content in whole frozen sections, and calculated losses during the
experimental procedure. Our estimate, 6 pg of total RNA per cell is
slightly lower than published estimates of 10 pg per
cell.15
Accordingly, 1000 laser-microdissected cells were
calculated to give 2 to 3 ng of total RNA when all losses during sample
preparation were considered.
Impact of T7 Amplification on Expression Profiles
The downside of all laser microdissection techniques is the labor
involved because the cells are cut out individually or in small
clusters. Time often sets a limit to the number of cells that can be
collected. Although microarray signal detection technology and labeling
methods develop rapidly, laser-microdissected material will require RNA
amplification during the foreseeable future. It is critical to achieve
sufficient amplification and yet maintain the expression profile. We
performed a series of experiments to determine how expression profiles
were affected by T7 amplification (Figure 3a)
. Ideally, the amplification should
preserve the relative abundance of all transcripts. However,
researchers are mostly interested in differences between samples, such
as changes in gene expression throughout a time-course, or comparisons
between a healthy state and a disease state. For these kinds of
analyses, even a biased amplification is acceptable if the bias is
reproducible and the differences are preserved. To address these
questions, we evaluated the effect of T7 amplification on 1) the
relative abundance of genes within each sample (preservation of
profiles), and 2) the difference in abundance between two samples
(preservation of ratios).
The expression analyses were consistently performed as two-color
experiments comparing two sets of RNA, and the results are presented in
ratio versus abundance (fluorescence intensity) plots. The
ratio (y axis) reflects the differential expression
for the given gene between the RNA sets, and the abundance
(x axis) reflects the mean expression intensity of
the gene. All values are displayed in log2 scale
for convenience. Briefly, total RNA was isolated from two different
sources, heart and kidney, and the gene expression was compared in
two-color microarray experiments. Approximately 403 genes were more
abundant in hearts, and 425 genes were more abundant in kidneys (Figure 3e
, P < 0.01). Next, 10 ng of total RNA from the heart
and kidney samples, respectively, were used for three rounds of T7
amplification. The gene expression between heart and kidney was again
compared in two-color experiments using the amplified material. The
approximate distribution of transcripts was preserved with 319 genes
being more abundant in hearts and 370 in kidneys (Figure 3f
,
P < 0.01). The experiments were repeated four times to
allow statistic analysis.
To evaluate how the amplification affected the relative abundance
within samples, expression profiles from nonamplified hearts were
compared to profiles from amplified hearts. Direct comparison in
two-color experiments was avoided because opposite strands, sense and
anti-sense RNA, are labeled in the nonamplified and T7-amplified
targets, respectively. Instead, the comparisons were made with heart
expression profiles obtained from different hybrizations. The
expression profiles were not preserved, which indicates that there
is a bias in the amplification (Figure 3b)
. The bias was not caused by
experimental variation, because the reproducibility of T7-amplified
profiles matched the reproducibility of nonamplified profiles (Figure 3, c and d)
. The signal intensity of microarray spots reflects gene
abundance, but also transcript length and uridine content because the
targets are labeled by incorporation of cyanine-conjugated dUTP. The
T7-amplification protocol includes priming with random hexamers, and
the transcript length is therefore systematically reduced, which could
affect the profile.
We next evaluated if the amplification preserved the differences
between samples: The relative abundance—the ratio of expression—of
each gene was compared between heart and kidney RNA before and after
amplification. The distribution of the genes that were expressed above
background is shown in a plot displaying nonamplified ratios on the
x axis and amplified ratios on the y axis (Figure 3g)
. The genes are distributed along a straight line (R = 0.84 for mean
ratios from four experiments), which clearly indicates that the
relative abundance of a given transcript between the two samples was
preserved through the amplification. The result confirms and extends
previously published data.10
A ratio versus
ratio plot displays no information about gene abundance. To evaluate
eventual effects of abundance, we divided the clones into 10 groups
based on their expression intensity, and calculated the Pearson
correlation coefficient (R) for each group separately (Figure 3h, A
to
T plots). Internal comparisons between repeated nonamplified (T to T
plots) and T7-amplified experiments (A to A plots) were also included.
The presented curves illustrate the impact of abundance on the
preservation of expression ratios (for details, see Material and
Methods). There was a striking dependence between gene abundance and
degree of correlation: for high-abundance genes, the correlation was
excellent with R approaching 0.90 for single experiments. For
low-abundance genes, the coefficients decreased and approached 0.2 at
intensities equivalent to background hybridization. Surprisingly, there
was no apparent difference between repetitions of nonamplified
experiments and amplified experiments. This strongly suggests that the
variability in the low-abundance region derives from the microarray
system and not from the amplification. The results even indicate that
further rounds of amplification might be applied if need be. Another
conclusion is that experiments need to be repeated many times to
correctly assess low-abundance expression ratios.
LMM and Microarray Hybridization of E9.5 Aorta
The first VSMCs appear around the dorsal aorta at
E9.5.16
The cells are initially confined to the ventral
and dorsal aspects of the vessel, but later occur around the full
circumference (see Figure 5, a and b
).17
These VSMCs
probably originate from the local mesenchyme that surrounds the
aorta.18
As a proof of principle, expression profiles were
generated from the endothelial cells and newly induced VSMCs of the
E9.5 aorta, and compared to profiles from mesenchymal cells located one
cell diameter further away from the vessel lumen (Figure 4
and Figure 5
). Approximately 50
cells of each category were captured from
one frozen section, and 20 sections were processed to accumulate the
1000 cells required for expression profiling. Three independent samples
from aorta and mesenchyme cells, respectively, were collected,
amplified, and hybridized to microarrays printed with 3800 randomly
selected cDNA clones. A section of a false color overlay of such a
hybridization is shown in Fig 6a
.
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displays 83 statistically
significant (P < 0.05) genes with
aorta/mesenchyme mean expression ratios >1.5-fold (highlighted genes
in Fig 6b
). The genes fall into broad range of categories that involve
most aspects of cell function. A striking number of genes belong to
groups with putative regulatory functions such as growth factors,
extracellular matrix components, signal transduction components, DNA
binding factors, and so forth. Eleven of the genes are previously known
vascular markers or markers for basement membranes,19
which confirms the accuracy of the profiles. Among the previously known
vascular markers are several genes with important regulatory functions;
Tie-2, endoglin, PDGFB, and integrin-ß1.
Mice that lack any of these genes fail to complete various aspects of
vascular development.19
|
. No genes with a previous record
as vascular, smooth muscle, or basement membrane markers are found in
the list.
|
Eleven genes were selected for validation with quantitative PCR. The analysis was performed on T7-amplified RNA from 1000 independently LMM/LPC dissected cells from E9.5 aorta and mesenchyme, respectively.
Two genes, MMP2 and tbc1, did not amplify at all,
probably because of insufficient optimization. The distribution of
transcripts for the other genes confirmed the results from the
microarray analyses (Table 3)
, with a
Pearson correlation coefficient of 0.71 for genes with <20-fold
overexpression. However, the PCR-quantified ratios for
endoglin, tie-2, and -SMA were much
higher than estimated from the array experiments, which illustrates a
predicted phenomenon: the endoglin and tie-2
array signal in the mesenchymal pool was not greater than the mean
yeast signal. Consequently, the measured ratios reflect the difference
between a significant signal in the aorta pool and background
hybridization in the mesenchymal pool. Quantitative PCR, on the other
hand, gives a true estimate of ratios even from rare transcripts
because the background signal generated with PCR is magnitudes lower.
The difference in -SMA ratio might stem from use of
slightly older embryos in the PCR experiment: the dorsal aortas were
captured at the very onset of -SMA expression, and even
slight variation in age between samples might increase or decrease the
number of -SMA-positive cells considerably. More
importantly, it has been shown that the border of -SMA
expression is diffuse initially with labeling of mesenchymal cells
further away from the aorta lumen.17
With time, the
expression becomes confined to the vascular wall cells. Alternatively,
there is cross-hybridization between actin paralogs on the array.
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Acknowledgements |
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Footnotes |
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Supported by the Association of International Cancer Research, The Swedish Cancer Foundation, the Inga-Britt and Arne Lundbergs Foundation, the Åke Wibergs Foundation, the Magnus Bergvalls Foundation, the Svenska Sällskapet för Medicinsk Forskning, and Göteborg University.
Accepted for publication November 14, 2001.
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References |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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 C. King, N. Guo, G. M. Frampton, N. P. Gerry, M. E. Lenburg, and C. L. Rosenberg Reliability and Reproducibility of Gene Expression Measurements Using Amplified RNA from Laser-Microdissected Primary Breast Tissue with Oligonucleotide Arrays J. Mol. Diagn., February 1, 2005; 7(1): 57 - 64. [Abstract] [Full Text] [PDF]
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C. Michel, C. Desdouets, B. Sacre-Salem, J.-C. Gautier, R. Roberts, and E. Boitier Liver Gene Expression Profiles of Rats Treated with Clofibric Acid: Comparison of Whole Liver and Laser Capture Microdissected Liver Am. J. Pathol., December 1, 2003; 163(6): 2191 - 2199. [Abstract] [Full Text]
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 Sun Hee Yim, J. M. Ward, Y. Dragan, A. Yamada, P. C. Scacheri, S. Kimura, and F. J. Gonzalez Microarray Analysis Using Amplified mRNA from Laser Capture Microdissection of Microscopic Hepatocellular Precancerous Lesions and Frozen Hepatocellular Carcinomas Reveals Unique and Consistent Gene Expression Profiles Toxicol Pathol, April 1, 2003; 31(3): 295 - 303. [Abstract] [PDF]
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C. Bondjers, M. Kalen, M. Hellstrom, S. J. Scheidl, A. Abramsson, O. Renner, P. Lindahl, H. Cho, J. Kehrl, and C. Betsholtz Transcription Profiling of Platelet-Derived Growth Factor-B-Deficient Mouse Embryos Identifies RGS5 as a Novel Marker for Pericytes and Vascular Smooth Muscle Cells Am. J. Pathol., March 1, 2003; 162(3): 721 - 729. [Abstract] [Full Text] [PDF]
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M. Takemoto, N. Asker, H. Gerhardt, A. Lundkvist, B. R. Johansson, Y. Saito, and C. Betsholtz A New Method for Large Scale Isolation of Kidney Glomeruli from Mice Am. J. Pathol., September 1, 2002; 161(3): 799 - 805. [Abstract] [Full Text] [PDF]
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