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Technical Advances |
From the Departments of Pathology*
and Internal
Medicine,
Justus-Liebig-University Giessen,
Giessen, Germany
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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10 ng.
This technique was used to investigate intrapulmonary arteries from
mouse lungs (500 cell equivalents). Using filters with 1176
spots, three independent experiments showed a high consistency
of expression for the preamplified cDNAs. These profiles differed
primarily from those of total lung homogenates. Additionally,
in experimental hypoxia-induced pulmonary hypertension,
amplified cDNA from intrapulmonary vessels of these lungs was compared
to cDNA from vessels dissected from normoxic lungs. Validation by an
alternative method was obtained by linking microdissection with
real-time polymerase chain reaction (PCR). As suggested by the array
data, nine selected genes with different factors of
up-regulation were fully confirmed by the PCR technique. Thus,
a rapid protocol is presented combining microdissection and array
profiling that demands low quantities of initial RNA to assess reliably
cell-type-specific gene regulation even within nonneoplastic complex
tissues.
To select cell types from complex tissues, microdissection techniques have been successfully used. Within a few years these techniques have become an accepted tool aiming to investigate complex tissues in a more detailed manner. They have been shown to isolate precisely single cells or cell clusters under optical control for use in several downstream applications for DNA, RNA, and protein analysis.5-7 In particular, the fragile mRNA can be obtained in a quality that is even suitable for construction of cDNA libraries or array hybridization.
As the amount of RNA from microdissected material is often limited and not sufficient for hybridization, a preamplification technique has to be incorporated. This ideally results in an accurate amplification of all RNAs, thus representing the original mRNA pool and preserving the expression profile. The T7-based linear amplification8,9 was the first approach to be shown to generate a representative mRNA profile that allows to combine microdissection with array technology.10,11 As the linear amplification is very time consuming and susceptible to various disturbances and even failures leading to RNA loss and degradation, a PCR-based technique was suggested.12 This approach uses the ability of reverse transcriptase to add nucleotides to the 3' end of the cDNA strand and allows a second primer to add. Thus, cDNAs with defined 5' and 3' ends for PCR amplification are obtained (commercially available as SMART PCR; Clontech, Palo Alto, CA).13 It was shown that high-, medium-, and low-abundance transcripts are amplified in a representative manner14 and that full-length cDNAs are provided.13,15 Moreover, application of SMART-amplified cDNA to arrays resulted in a very high homology of the expression profile of several hundred genes when compared to unamplified cDNA16,17 indicating that the relative proportions of the mRNAs were maintained.
In intact mouse lungs, originating from control animals and those with pulmonary hypertension because of chronic hypoxia, we aimed to establish a rapid and reproducible protocol that allows a reliable mRNA profiling of intrapulmonary arteries containing a limited amount of microdissected cell profiles. Hereby, the application to nonneoplastic tissue required a special effort in precision because message regulation often varies in a smaller extent compared to tumors. Laser-assisted microdissection was used for isolation of the vessels that represent only a minor portion of the lung tissue. Using SMART PCR for preamplification, we modified the protocol to introduce the entire cDNA to PCR amplification and subsequent array hybridization. To validate differential gene expression measured by cDNA arrays, the results were verified by a second independent technique based on microdissection in combination with relative mRNA quantitation by real-time PCR.18
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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All animal experiments were approved by the local authorities (Regierungspräsidium Giessen, no. II25.3-19c20-151 GI20/10-Nr.22/2000). Male BALB/cAnNCrlBR mice (20 to 22 g; Charles River, Sulzfeld, Germany) were exposed to normobaric hypoxia [inspiratory O2 fraction (FiO2) 0.10] in a ventilated chamber. The level of hypoxia was held constant by an autoregulatory control unit (O2 controller model 4010; Labotect, Göttingen, Germany), supplying either nitrogen or oxygen. Excess humidity in the recirculating system was prevented by condensation in a cooling system. CO2 was continuously removed by soda lime. Mice exposed to normobaric normoxia were kept in a similar chamber at an FiO2 of 0.21. After 21 days, the animals were intraperitoneally anesthetized with 180 mg of sodium pentobarbital/kg body weight. A cannula was inserted into the trachea by tracheostomy, a midline sternotomy was performed, and the lungs were flushed via a catheter in the pulmonary artery with Krebs Henseleit buffer (125.0 mmol/L NaCl, 4.3 mmol/L KCl, 1.1 mmol/L KH2PO4, 2.4 mmol/L CaCl2, 1.3 mmol/L MgCl2, and 13.32 mmol/L glucose) at a pressure of 20 cm H2O at room temperature. It was equilibrated with a gas mixture of 1% O2, 5.3% CO2, balanced N2. NaHCO3 was adjusted to result in a constant pH range of 7.37 to 7.40. During perfusion of the lungs the buffer was allowed to drain freely from a catheter in the left ventricle. Once the effluent was clear of blood, 800 µl of prewarmed TissueTek (Sakura Finetek, Zoeterwoude, The Netherlands) were instilled into the airways via the tracheal cannula. After ligation of the trachea, the lungs were excised and immediately frozen in liquid nitrogen. Preparation of the hypoxic animals was continuously performed in the hypoxic environment. The right ventricular wall was trimmed from the left ventricle plus septum to calculate the ratio of right ventricle wall/(left ventricle plus septum) of the dried heart tissue. Right heart hypertrophy after 21 days of hypoxia was additionally ascertained by separate experiments including normoxic and hypoxic animals. Data were compared by a paired t-test.
Isolated Lung Perfusion and Measurement of Pulmonary Artery Pressure
In separate experiments pulmonary artery pressure was measured in
an in situ isolated lung preparation. Mice were deeply
anesthetized intraperitoneally with sodium pentobarbital and
anti-coagulated with heparin (1000 U/kg) by intravenous injection.
After placing on a heated table (37°C), animals were intubated via
tracheostoma and ventilated with room air (300 µl tidal volume, 90
breaths/min, and 3 cmH2O positive end-expiratory
pressure). Midsternal thoracotomy was followed by insertion of
catheters into the pulmonary artery and left atrium. Using a
peristaltic pump (ISM834A V2.10; Ismatec, Glattbrugg, Switzerland),
buffer perfusion via the pulmonary artery was started at 4°C and a
flow of 0.2 ml/min. The buffer contained 120 mmol/L NaCl, 4.3 mmol/L
KCl, 1.1 mmol/L KH2PO4, 2.4
mmol/L CaCl2, 1.3 mmol/L
MgCl2, and 2.4 g/L of glucose as well as 5%
(w/v) hydroxyethylamylopectin (molecular weight, 200,000).
NaHCO3 was adjusted to result in a constant pH
range of 7.37 to 7.40. In parallel with the onset of artificial
perfusion, ventilation was changed from room air to a mixture of 5.3%
CO2, 21.0% O2, balanced
N2. After rinsing the lungs with 
20 ml of
buffer, the perfusion circuit was closed for recirculation (total
system volume, 13 ml) and left atrial pressure was set at 2.0 mm Hg.
Meanwhile, the flow was slowly increased from 0.2 to 2 ml/min and the
entire system heated to 37°C. Pressures in pulmonary artery and left
atrium were registered via transducers. The given pulmonary artery
pressure values were taken after an initial steady state period of 20
minutes. Data were recorded from normoxic mice and from mice after 21
days of chronic hypoxia and analyzed by a paired t-test.
Laser-Assisted Microdissection
Microdissection was performed as described in detail
previously.19,20
In brief, cryosections (6 µm) from lung
tissue were mounted on glass slides. After hemalaun staining for 45
seconds, the sections were subsequently immersed in 70% and 96%
ethanol and stored in 100% ethanol until use. Not more than 10
sections were prepared at once to restrict the storage time.
Intrapulmonary arteries with a diameter of 250 to 500 µm were
selected and microdissected under optical control using the Laser
Microbeam System (P.A.L.M., Bernried, Germany). Afterward, the vessel
profiles were isolated by a sterile 30-gauge needle. Needles with
adherent vessels were transferred into a reaction tube containing 200
µl of RNA lysis buffer (Figure 1)
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Lysis buffer for mRNA extraction consisted of 4 mol/L of guauidine thiocyanate (GTC), 25 mmol/L of Na3 citrate, 0.5% sarcosyl, and 0.72% ß-mercaptoethanol. After incubation for 10 minutes at room temperature, 20 µl of 2 mol/L NaAc, 220 µl of phenol (pH 4.3), and 60 µl of chloroform/isoamylalcohol (24:1) were added. The samples were vortexed and centrifuged for 15 minutes at 4°C. The aqueous layer was collected, 1 µl of glycogen (1 mg/ml) added, and afterward precipitated with 200 µl of isopropanol. Samples were frozen for 1 hour at -20°C and centrifuged for 15 minutes. The pellets were washed with 75% ethanol and air-dried. After resuspension in 10 µl of H20, DNase digestion (1 U, 30 minutes, 37°C; Ambion, Austin, TX) was performed. Afterward, extraction was repeated and RNA was diluted in 4 µl of H2O.
cDNA Synthesis and Amplification
Total RNA was reverse-transcribed using the SMART PCR cDNA Synthesis Kit (Clontech) with slight modifications. Four µl of total RNA, 1 µl of cDNA Synthesis (CDS) Primer (diluted to a concentration of 5 µmol/L), and 1 µl of SMART II oligonucleotide (diluted to a concentration of 5 µmol/L) were mixed and incubated at 70°C for 8 minutes. After short spinning, 2 minutes on ice and 2 minutes at 42°C, a master mix containing 2 µl of 5x buffer, 1 µl dithiothreitol (20 mmol/L), 1 µl dNTP (10 mmol/L) and 0.5 µl RNase H- Moloney murine leukemia virus reverse transcriptase (PowerScript, Clontech) was added and incubated at 42°C for 1 hour. Afterward, cDNA was mixed with 38.5 µl of TE buffer (10 mmol/L Tris, pH 7.6, 1 mmol/L ethylenediaminetetraacetic acid) and purified by the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Therefore, 250 µl of buffer PB were added to the cDNA to load a column. According to the manufacturer‘s protocol, the columns were washed once. For elution, 45 µl of elution buffer (10 mmol/L Tris, pH 8.5) were applied to the center of the column, incubated for 2 minutes, and centrifuged. To improve the recovery, this step was repeated using the first eluate again.
From the eluted cDNA (44 µl), 2 µl were separated for further
determination of the amplification factor. For the PCR-based
amplification, the remaining 42 µl of cDNA were mixed with 5 µl of
10x buffer, 1 µl PCR primer (10 µmol/L), 1 µl dNTP (10 mmol/L),
and 1 µl Advantage 2 polymerase mix. PCR conditions were
95°C for 1 minute, followed by 19 cycles with 95°C for 15 seconds,
65°C for 30 seconds, and 68°C for 3 minutes. The resulting PCR
product was purified using the QIAquick columns as described above.
Forty-four µl of elution buffer were applied twice for elution and 2
µl were separated for determination of the amplification factor. All
incubations were performed with a GeneAmp 2400 PCR cycler (PE Applied
Biosystems, Foster City, CA).
Probe Labeling and Array Hybridization
For array hybridization, we used nylon filters with 1176 spotted
cDNA (Mouse 1.2 II Atlas cDNA Arrays, Clontech). The purified PCR
product was labeled with 
-32P dATP using the
Atlas SMART Probe Amplification Kit (Clontech). Forty-two µl of PCR
product and 1 µl of CDS primer were heated at 95°C for 8 minutes.
After 2 minutes at 50°C, a master mix containing 6 µl of 10x
labeling buffer, 5 µl of dNTP (without dATP), 4 µl of
-32P dATP (Amersham Pharmacia Biotech,
Freiburg, Germany), and 1 µl of Klenow enzyme were added, mixed, and
incubated for 30 minutes. Reaction was stopped by applying 2 µl of
0.5-mol/L ethylenediaminetetraacetic acid. Labeled DNA was purified by
QIAquick columns as described above, eluted twice with 100 µl of
elution buffer, and resulted in 5 to 8 x
106
cpm. Afterward, array hybridization was
performed according to the protocol. Filters with
32P-labeled PCR product were incubated at 68°C
overnight. They were washed three times in 500 ml of 2x standard
saline citrate and 1% sodium dodecyl sulfate at 68°C for 30 minutes.
Finally, they were wrapped in plastic and exposed to an imaging plate
(Fuji Photo Film, Tokyo, Japan) in lead sheathing. The film was read
with a phosphorimaging system (BAS RPI 1000, Fuji Photo Film).
Array Analysis
Analysis was performed using the AtlasImage 1.5/2.0 software (Clontech). Global normalization was calculated by the sum method. For both arrays, differences of signal intensity minus background were added for all values over background. Afterward, normalization coefficient was determined by calculating the ratio of array 1 sum and array 2 sum. After normalization, background was substracted, ratio threshold was set at 2, and difference threshold was set at 550.
Determination of the Amplification Factor
Based on the following equation, we used comparative quantitation
(
CT). Real-time PCR was performed by the
Sequence Detection System 7700 (PE Applied Biosystems).
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Applying either 2 µl of nonamplified cDNA or 2 µl of amplified PCR
product, 25 µl Universal Master Mix (Applied Biosystems),
porphobilinogen deaminase (PBGD) forward primer and reverse primer
(Table 1)
in a final concentration of 900
nmol/L and hybridization probe (Table 1)
in a final concentration of
200 nmol/L were mixed in an end volume of 50 µl. Cycling conditions
were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15
seconds, and 61°C for 60 seconds.
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, cyclooxygenase 2 (COX2), and tissue factor.
Preparation and PCR conditions were identical to those described
before, primer/probe sets are given elsewhere.21,22 Relative mRNA Quantitation
The above-mentioned equation was also used for relative mRNA
quantitation by real-time PCR. The target gene was normalized to an
internal standard gene. Therefore, PBGD mRNA was used, an ubiquitously
as well as consistently expressed standard gene that is free of
pseudogenes. For cDNA synthesis, reagents and incubation steps were
applied as described.18
The reactions were set up with the
SYBR Green PCR Core Reagents (Applied Biosystems) according to the
manufacturer‘s protocol. Using the oligonucleotide primer pairs given
in Table 1
, for each gene 1 µl of the concerning primers (final
concentration, 200 nmol/L) and 2 µl of cDNA were added to a final
volume of 50 µl. Cycling conditions were 95°C for 6 minutes,
followed by 45 cycles of 95°C for 20 seconds, 58°C for 30 seconds,
and 73°C for 30 seconds. Because of the nonselective dsDNA binding of
the SYBR Green, gel electrophoresis was performed to confirm the
exclusive amplification of the expected PCR product.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To investigate the preservation of the mRNA expression profile
during the cDNA preamplification procedure, extracted mRNA from lungs
treated with lipopolysaccharide and
interferon-
20
was diluted to amounts
comparable to microdissected material. Afterward, preamplification was
performed using SMART PCR. The generated PCR product as well as the
unamplified cDNA then underwent real-time quantitative PCR. To test the
impact of preamplification on different amounts of initial mRNA copy
numbers, four genes were selected representing different levels of mRNA
expression. Tumor necrosis factor- was seen to be a highly expressed
gene after lipopolysaccharide/interferon- stimulation, PBGD and COX2
were moderately expressed, and tissue factor expression was very low.
When comparing the ratios of cDNA amplified by 15 cycles to
preamplified status, consistency of the amplification step for all
genes was noted (Table 2)
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Microdissection of lung vessels was performed to isolate 30 to 40
vessel profiles corresponding to 500 cell equivalents (Figure 1)
.
RNA was extracted, transcribed to cDNA, and applied to PCR-based
amplification. In preliminary experiments undertaken for the gene that
first reached the plateau phase (PBGD), we noted that amplification of
more than 22 cycles did not result in a further increase of PCR
product. Thus, we stopped the preamplification after cycle 19 to
ascertain analytic conditions within the exponential phase of PCR.
After RNA extraction of the microdissected vessels, we routinely
determined the factor of amplification obtained by SMART PCR.
Therefore, 2 µl of cDNA were separated from the purified cDNA (=1/22
of the original cDNA) as were 2 µl of the purified DNA (1/22 of total
PCR product) after 19 PCR cycles. Both were subjected to real-time PCR.
The mean threshold cycle of the original purified cDNA amounted to
33.68 ± 0.37 (mean ± SEM; n = 14).
Comparing the threshold cycle after amplification to the nonamplified
cDNA, an amplification factor of 882 ± 144 (mean ± SEM;
n = 14) was calculated for PBGD mRNA.
Real-time PCR for PBGD mRNA also allowed assessment of the full-length
transcription of this mRNA and thus predicted the quality and integrity
of multiplied DNA. In the used primer/probe system (Table 1)
one primer
was positioned in exon 1 at the start codon so that only completely
transcribed mRNA is measured.
Array Analysis 1: Expression Profile Comparison of Homogenized Lung Tissue versus Microdissected Vessels
First, RNA from homogenized lung tissue as well as
PCR-preamplified cDNA from microdissected vessels were applied to
prespotted membranes. The expression profiles were found to differ
completely (Figure 2)
. Although the
filter with vessel RNA showed spots that are weak or nondetectable on
the homogenate array (ie, cardial/slow skeletal troponin C, cardiac
troponin T2, non-muscle myosin light chain), several strong spots of
the homogenate array were much weaker on the isolated vessel array (ie,
68-kd DNA polymerase 2, GAPDH).
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Three independent hybridization experiments were performed with microdissected vessels from two different normoxic lungs to assess the reproducibility of the technique. Normalization was calculated by the sum method, afterward the background was subtracted. On filter A, 237 spots were detectable at least twice over the background (20.2%), filter B exhibited 266 spots (22.6%), and filter C exhibited 228 spots (19.4%). Comparing filter A to B, 192 spots were common, corresponding to 81.0% of A and 72.2% of B. Comparing filter A to C, 175 spots were common, corresponding to 73.8% of A and 76.8% of C. Thus, high consistency of the expression profile among different microdissected vessels was noted.
Array Analysis 3: Comparison of Microdissected Vessels; Normoxia versus Hypoxia
Within 21 days of chronic hypoxia a significant increase in the
ratio of the right ventricular wall/left ventricle plus septum from
0.31 ± 0.01 to 0.44 ± 0.02 (mean ± SEM,
n = 9 each, P < 0.0002) was induced as
compared to normoxic controls. Pulmonary artery pressure, measured in
the isolated perfused lung preparation increased from 8.5 ± 0.2
in normoxic mice to 11.1 ± 0.2 after chronic hypoxia (mean
± SEM, n = 6 each, P < 0.006). In
accordance, a remodeling process effecting a thickening of the vessel
wall with proliferation of smooth muscle cells was seen (Figure 3)
. To investigate differential gene
expression, vessels from the two mouse lungs that were kept in normoxia
were opposed to vessels from two mouse lungs that were kept in hypoxia.
Three independent hybridization experiments were performed. Overall,
the expression profiles were very similar between normoxic and hypoxic
vascular tissue. In the example of hypoxic vessels demonstrated in
Figure 4
, 207 spots (=17.6%) were
detected at least two times higher than the background. One hundred
seventy-five of these spots were common with those detected in the
microdissected vessels of a normoxic lung analyzed in parallel.
Twenty-two cDNA signals differed to an extent of the hypoxia/normoxia
ratio of <0.5 or >2.0. Among those, 14 genes were up-regulated and 8
down-regulated. Corresponding results were obtained for the second
hypoxic lung undergoing cDNA array hybridization.
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1), procollagen 1 (subunit 2), procollagen 3 (subunit
1), carbonic anhydrase 3, matrix -carboxyglutamate protein,
non-muscle myosin light chain 3, serine proteinase inhibitor 3,
cytochrome b-245 ( polypeptide), and AXL receptor tyrosine kinase.
The factors of up-regulation in response to hypoxia measured in the
three experiments are given in Table 3
.
The mean induction factor varied between 1.22 and 5.43.
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RNA was extracted from microdissected vessels of the same lungs.
Applying SYBR Green and specific primers for the nine genes, mRNA was
quantified in three independent experiments each. Normalized to the
standard gene PBGD mRNA, we found an up-regulation of the genes that
matched very well the array results (Table 3)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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10 to 50 per working cycle, it has to be repeated two or
three times, including several incubation and cleaning steps, thereby
exposing the RNA to the danger of loss and degradation. In our hands,
the profile of the mRNA pool was maintained when using the T7-based
technique, but the mean amplification factor ranged far below that
described for the PCR-based technique in this approach, especially when
introducing small amounts of original RNA. The currently used PCR-based amplification technique (SMART, Clontech) has already been shown to result in confidential amplification of the RNA pool.14,16,17 Using four selected genes representing different levels of RNA copy numbers, we confirmed this observation demonstrating preservation of the mRNA profile by real-time quantitative PCR. Thus, the SMART PCR is suitable for efficient amplification with sufficient accuracy.
The successful combination of PCR-based cDNA preamplification and array
hybridization has been described several times.16,17,25
Leethanakul and colleagues26
applied the technique to
microdissected material. Collecting 5000 cells, only a minor portion
of the cDNA could be introduced to PCR amplification. This procedure
corresponds to the protocol provided by the manufacturer (Probe
amplification kit, Clontech), which recommends to amplify only
one-tenth of the initially transcribed RNA, most probably because of an
inhibitory effect of the added cDNA primers during the following PCR
amplification. Indeed, the attempt to apply a larger fraction of cDNA
to the PCR resulted in a loss of amplification efficiency when measured
by real-time PCR (data not given). Thus, we modified the protocol by
introduction of a purification step after cDNA synthesis. Without
significant loss, this allows the separation of the disturbing primer
fraction from the cDNA and the total amount of cDNA may then be applied
to the subsequent preamplification. After SMART PCR, the product is
cleaned again and completely introduced to the probe labeling process,
guaranteeing that the total amount of initial RNA is applied to the
hybridization reaction.
To assess the power of amplification, both the initial cDNA and the amplified PCR product were routinely measured by real-time PCR. In addition, knowledge of the absolute threshold cycle for PBGD mRNA enabled us to foresee the success of the hybridization procedure. Obtaining a threshold cycle earlier than 26 for amplified cDNA, the product was sufficient to result in a good hybridization signal. Using this modified protocol, the PCR-based preamplification could be performed much faster than the linear T7-based technique and resulted reliably in a suitable quality and quantity of amplified cDNA for hybridization.
When aiming to generate cDNA profiles representative for the input mRNA it is crucial to stop the PCR cycling before the first gene reaches its plateau phase. Because the expression profile would inevitably change, the timely termination has to be fixed in preliminary experiments. Although we noted plateauing after 22 cycles of amplification for PBGD, we stopped the PCR already after 19 cycles to be safely within the exponential phase. The amount of cycles may vary and can be adapted, potentially increasing the factor of amplification.
For SMART amplification the quality of RNA is crucial for its success.
Using our microdissection protocol, the time of potential RNA
degradation was minimized. To assess the quality of mRNA especially
after amplification, our PBGD primer/probe system for real-time PCR is
considered to be a good measure. With a length of 1100
bp,27
the PBGD mRNA belongs to the medium-length RNA
fraction and might be representative for the RNA pool. Because only
completely transcribed and amplified PBGD cDNA copies are detected, it
allows estimation of the shape of the mRNA pool.
To test our protocol, we selected lung vessels for microdissection. The
laser was drawn along the tunica adventitia to comprise the gene
expression profile of tunica media and intima in total. However, to
increase the specificity, the thin endothelial layer may be separated
from the tunica media by the laser beam so that both structures can be
analyzed separately. Thirty to 40 vessel sections were collected with a
diameter of 250 to 500 µm. Smooth muscle cells represent a cell type
bearing only a minor amount of mRNA per cell. In total, we forwarded
mRNA from 500 cell equivalents to the array analysis. Based on the
calculated amplification factor of 1000 on the one hand and the
necessity to apply 5 to 10 µg total RNA to the array for a good
signal on the other hand, 10 ng of extracted RNA after
microdissection are requested. Because most cell types contain 20 to 40
pg of total RNA per cell, the overall quantity of introduced cells into
the analytical procedure is easily calculated. As most of the applied
arrays need similar amounts of total RNA for hybridization the
described approach is correspondingly applicable to the diverse
available array systems. We used nylon filters to take advantage of an
increased sensitivity because of the use of radioactive labeled cDNA
(32P). In addition, the membranes can be stripped
and reused for at least three times thus reducing costs.
As vessels represent only a minor fraction of the lung tissue we expected a different expression profile of the homogenized lung tissue compared to the isolated vessels. Indeed, the pattern of the homogenate differed primarily from that of the microdissected lung arteries. Addressing the regulation of gene expression of defined structures and/or specific cell types within complex tissues, the microdissection is an indispensable precondition to generate valid data. The consistency of the expression profile noted on analysis of microdissected vessels from independent experiments supports the validity and reproducibility of this approach.
Chronic hypoxia regularly induces an increase in pulmonary artery
pressure and provokes remodeling of the vasculature such as the
proliferation of smooth muscle cells and thickening of the arterial
tunica media. Therefore, the chronic hypoxia represents a common model
to investigate pulmonary hypertension. After 21 days of chronic hypoxia
in mice, increases in the relative right ventricular weight and
thickness of the vessel walls were detectable suggesting up-regulation
of genes related to structural growth. Of interest, extracellular
matrix components such as procollagens have already been shown to be
up-regulated during the remodeling process of pulmonary arteries. Berg
and colleagues28
found elevated mRNA levels for
procollagen 4 2, 1 1, and 3 1 regulated in response to
short-term and long-term hypoxia. In the context of primary pulmonary
hypertension, Botney and colleagues29
described the
detection of procollagen 1 1 in the tunica media and neointima
of small muscular arteries whereas normal lung vessels were negative.
Thereby, the increased extracellular matrix synthesis might be induced
by transforming growth factor-ß subtypes.30
Apart from the new chances and possibilities opened by array technology, many voices warn against potential pitfalls.31 These pitfalls concern correct assignment of the spotted oligonucleotides or cDNAs, mistakes during array analysis (ie, wrong evaluation of normalization parameters), as well as measurement of smear and false-positive spots. Additionally, although the SMART amplification may well preserve the representation of most of the original RNAs, exceptions may be possible.14 To exclude these mistakes it is advisable to check the results by an independent approach. To this end, Sgroi and colleagues23 suggested the use of real-time PCR. Following this line, we verified differential expression derived from the array analysis by our established combination of microdissection and mRNA quantitation applying real-time PCR. Comparison of the selected genes’ mRNA to the standard gene PBGD mRNA is used for normalization of the technique. The ratio of relative expression in hypoxic versus normoxic vessels is equivalent to the factor of hypoxic induction. These factors of hypoxia-induced gene up-regulation matched very well with the array data. Even if a factor less than 2 is not regarded to be significant as noted for some genes of interest, both techniques resulted in a similar ratio.
In conclusion, we present an optimized, rapid, and reproducible
strategy to combine laser-assisted microdissection of 500 cell
equivalents with array hybridization. Modification of the protocol of
PCR-based SMART preamplification allows the introduction of the total
amount of the extracted RNA from microdissected cells. To test the
validity of the technique, real-time PCR was used as an alternative
method to confirm the differential gene expression that was seen by
array comparison. Apart from tumor-specific analysis, gene regulation
even within nonneoplastic complex tissues and intact organs can be
determined in a cell-type-specific manner. Thus, the presently
described method combines the advantages of microdissection with the
advantages of a broad transcriptome analysis by cDNA arrays.
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Acknowledgements |
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Footnotes |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 547, project Z1.
L. F. and S. K. contributed equally to these studies.
Accepted for publication September 25, 2001.
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 S. A. Gharib, D. L. Luchtel, D. K. Madtes, and R. W. Glenny Global gene annotation analysis and transcriptional profiling identify key biological modules in hypoxic pulmonary hypertension Physiol Genomics, June 16, 2005; 22(1): 14 - 23. [Abstract] [Full Text] [PDF]
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 N. Baumgarth, R. Szubin, G. M. Dolganov, M. R. Watnik, D. Greenspan, M. Da Costa, J. M. Palefsky, R. Jordan, M. Roederer, and J. S. Greenspan Highly Tissue Substructure-Specific Effects of Human Papilloma Virus in Mucosa of HIV-Infected Patients Revealed by Laser-Dissection Microscopy-Assisted Gene Expression Profiling Am. J. Pathol., September 1, 2004; 165(3): 707 - 718. [Abstract] [Full Text] [PDF]
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C. E. Rogler, T. Tchaikovskaya, R. Norel, A. Massimi, C. Plescia, E. Rubashevsky, P. Siebert, and L. E. Rogler RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples Nucleic Acids Res., August 25, 2004; 32(15): e120 - e120. [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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