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Articles |
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Department of Pediatric Oncology/Hematology, Charité Medical Center, Campus Virchow-Klinikum, Humboldt-University at Berlin, 13353 Berlin, Germany.
a Address correspondence to this author at: Otto-Heubner-Centrum für Kinder- und Jugendmedizin der Charité, Campus Virchow-Klinikum, Forschungshaus R 2.0412, Augustenburger Platz 1, 13353 Berlin, Germany. Fax 49-30-450-59911; e-mail
sven.wellmann{at}charite.de.
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Abstract |
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Methods: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to ß2-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.
Results: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF121 and VEGF165 accounted for 94% (SD = 7.6%) and VEGF189 only 6% (SD = 3.7%).
Conclusions: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF is a 34- to 42-kDa heparin-binding, dimeric, disulfide-bound glycoprotein existing in at least five homodimeric isoforms. The monomers consist of 121, 145, 165, 189 and 206 amino acids (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206), respectively (15)(16). The precise functional differences among the isoforms are not yet known. Differences in solubility, receptor affinity, and mitogenic potency have been described [reviewed in Neufeld et al. (17)]. The primary VEGF transcript derives from a single VEGF gene coding for eight exons. Variable alternative mRNA splicing involves exons 6 and 7, whereas the amino acids encoded by exons 1–5 and 8 are conserved in all isoforms. Exons 6 and 7 encode two distinct heparin-binding domains. The presence or absence of these domains influences solubility and receptor binding. The heparin-binding domain encoded by exon 6 determines binding to the extracellular matrix. Thus, isoforms containing the domain encoded by exon 6 (VEGF145, VEGF189, and VEGF206) are bound tightly to cell surface heparin-containing proteoglycans in the extracellular matrix (18)(19), whereas isoforms lacking the domain encoded by exon 6 are diffusible. VEGF165, which contains only one heparin-binding region encoded by exon 7, is moderately diffusible, whereas VEGF121, which lacks the domains encoded by exons 6 and 7, is highly diffusible.
At present, little is known on the relative abundance of the different VEGF splice variants in healthy cells of different origin and in the malignant counterparts. Whereas the major splice variants VEGF121, VEGF165, and VEGF189 are expressed in nearly all investigated tissues in different ratios, the variants VEGF145 and VEGF206 (20) as well as the recently described variants VEGF148 (21) and VEGF183 (22) are restricted to only a very few cell types and seem to be of minor importance.
In recent investigations based on competitive reverse transcription (RT)-PCR assays, the increased expression of VEGF165 and VEGF189 in osteosarcoma (23), renal cell carcinoma (24), non-small-cell lung cancer (25), and colon cancer (26) was correlated with neovascularization, tumor progression, and poor prognosis. Cheung et al. (27) pointed out that during malignant progression, an angiogenic switch favoring the variants that encode for the shorter diffusible isoforms (VEGF121 and VEGF165) occurs. Furthermore, Cheng et al. (28) could show that intracerebral tumor-associated hemorrhage is caused by overexpression of the variants that encode for the short diffusible isoforms, VEGF121 and VEGF165. On the other hand, the cell-associated isoform VEGF189 contributes to successful xenotransplantability of various human solid tumors via augmentation of stromal vascularization (29).
In this study, a novel real-time RT-PCR was developed for specific quantification of VEGF splice variants to investigate the expression of VEGF splice variants and their ratio in healthy and malignant tissues, using the new LightCycler technology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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quantitative real-time rt-pcr
Primer and probe design.
Quantification of the mRNA message
coding for VEGF121,
VEGF165, VEGF189, and
VEGFtotal was performed using LightCycler
technology (Roche Diagnostics). To reach high conformity of the PCR
kinetics for the VEGF splice variants and
VEGFtotal, a common forward primer, ex3fo
(5'-CCCTGATGAGATCGAGTACATCTT-3'), and a common fluorescent
hydrolyzation probe, VEGF-probe (5'-ATCCTGTGTGCCCCTGATGCGATGCGGT-3'),
both located in exon 3 (part of the conserved region of all
VEGF splice variants) were designed (Fig. 1
). Specific amplification of each splice variant was performed
exclusively with specific reverse primers spanning the variant specific
exon boundaries: ex5/8re (5'-GCCTCGGCTTGTCACATTTT-3', 254-bp amplicon)
for VEGF121, spanning the boundaries of
exons 5 and 8; ex5/7re (5'-AGCAAGGCCCACAGGGATTT-3', 254-bp amplicon)
for VEGF165, spanning the boundaries of
exons 5 and 7; and ex6re (5'-AACGCTCCAGGACTTATACCG-3', 310-bp amplicon)
for VEGF189, located in exon 6. For
quantification of the complete VEGF message
(VEGFtotal), the common forward primer, the
common fluorescent probe, and a common reverse primer located in exon 8
(ex8re: 5'-ACCGCCTCGGCTTGTCAC-3') were used for simultaneous
amplification of all variants by external PCR. For normalization, the
published B2M primer/probe set (31) was adapted
to our conditions: B2M forward primer
(5'-GATGAGTATGCCTGCCGTGTG-3') and B2M reverse primer
(5'-CAATCCAAATGCGGCATCT-3'), which produce a 114-bp amplicon; and
B2M hydrolyzation probe
(5'-CCTCCATGATGCTGCTTACATGTCTCGATCCC-3'). Hydrolyzation probes were
labeled with a reporter dye (6-carboxy-fluorescein phosphoramidite) at
the 5' end and a quencher dye (5-carboxy-tetramethylrhodamine) at the
3' end. The similar calculated melting temperatures for all primers
enabled the use of the same cycling program for all samples. The
melting temperatures for primers and probes were calculated using OLIGO
5.0 (MedProbe) or the JAVA OLIGO program (TIB Molbiol).
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PCR conditions.
Quantitative PCR was performed in a total
reaction volume of 20 µL per capillary for the LightCycler format.
For conventional PCR, the amounts of reagents were scaled up to 30
µL. Bovine serum albumin (Sigma) was added only to the LightCycler
reaction mixture to avoid nonspecific binding of reagents to the
LightCycler glass capillary. The 20-µL reaction mixture contained
0.75 U of a temperature-release Taq DNA polymerase (Platinum DNA
Polymerase; Gibco BRL, Life Technologies), 2 µL of the supplied 10x
PCR buffer, 7.5 mM MgCl2, 0.2 mM dNTPs (Gibco
BRL, Life Technologies), 3 µg of bovine serum albumin (Sigma), 0.25
µM each primer, 0.1 µM fluorescent probe (primers and probes; TIB
Molbiol), and ± 0.4 ng of cDNA. The amplification conditions for
LightCycler consisted of an initial 1.5-min denaturation step at
95 °C, followed by 40 cycles of denaturation at 95 °C for 8
s, annealing at 68 °C for 12 s, and extension at 72 °C for
20 s. Conventional PCR cycling conditions were as follows: 3-min
denaturation step at 95 °C, followed by 40 cycles of denaturation at
95 °C for 15 s, annealing at 68 °C for 15 s, and
extension at 72 °C for 25 s.
Samples.
Fourteen cDNA samples from seven healthy and seven
malignant tissues (Clontech Laboratories) were investigated by
quantitative real-time PCR to demonstrate the applicability of the new
method. Healthy tissues were pooled from several healthy Caucasians:
lung, pancreas, kidney, liver, heart, skeletal muscle, and placenta.
Malignant tissues were obtained from well-characterized tumors that had
been explanted and propagated as xenografts in athymic nude mice, and
used as model systems for cancer research: tumor cell lines LuCa1, a
lung carcinoma explanted from a metastasis; LuCa2, a lung carcinoma
explanted from a tumor; PaCa, a pancreatic adenocarcinoma; BrCa, a
breast carcinoma; OvCa, an ovarian carcinoma; PrCa, a prostatic
adenocarcinoma; and CoCa, a colon adenocarcinoma. Each provided cDNA
preparation (± 0.2 ng/µL) was diluted 1:1 in water, and 4 µL of
this dilutions was used per 20-µL real-time PCR reaction volume.
Normalization and quantification.
To determine the absolute
copy number of the target transcripts, the cloned plasmid cDNAs for
B2M and for the VEGF mRNA splice variants
VEGF121,
VEGF165, and
VEGF189 were used to generate a calibration
curve. Purified plasmid cDNA templates were measured in a photometer,
and copy numbers were calculated from the absorbance at 260 nm
(A260). Plasmid cDNA was serially
diluted in log steps from 108 copies down to 10
copies in a 1-µL volume. A calibration curve was created by plotting
the threshold cycle (Ct) vs the known copy number for each plasmid
template in the dilutions. The copy numbers for all unknown samples
were determined by LightCycler software 3.1, according to the
calibration curve. To correct for differences in both RNA quality and
quantity between samples, data were normalized by dividing the copy
number of the target cDNA by the copy number of B2M.
Quantitative results are presented as copies of target gene per 1000
copies of B2M. For each tissue, B2M and the
target genes VEGF121,
VEGF165,
VEGF189, and
VEGFtotal were quantified simultaneously in
triplicate in one LightCycler run, together with dilutions of
calibrators spanning four orders of magnitude from 4 x
106 to 4 x 102 copies
per reaction mixture in duplicates and the appropriate non-template
controls for each PCR. The range from 4 x
106 to 4 x 102 copies
(corresponding to cycles 20–35) was chosen because the Cts for all
measured target copies were within ± 2 cycles of this range.
To have comparable conditions for analyzing VEGF expression in all 14 tissues, we set the mean Ct value of the first dilution of the calibrators (4 x 106) to cycle 20 by adjusting the baseline. Statistics were performed using Excel 97 computer software (Microsoft Inc.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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specificity
Specific detection of different VEGF variants was
achieved with the following primer pairs: for
VEGF121, primer pair ex3fo-ex5/8re; for
VEGF165, primer pair ex3fo-ex5/7re; and for
VEGF189, primer pair ex3fo-ex6re. For the
variants VEGF121,
VEGF165, and
VEGF189, 105 plasmid
copies were detectable with the appropriate primer sets with a mean Ct
of 24. Real-time RT-PCR with the "nonspecific" primer set for the
same copy number did not generate any reporter fluorescence signal even
after 40 PCR cycles, and did not show any PCR product in 3% agarose
gel electrophoresis after ethidium bromide staining (Fig. 2
).
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determination of pcr sensitivity and efficiency
The sensitivity of the PCR method using different primer/probe
sets was determined from the Ct values obtained with known quantities
of plasmid cDNA. All calibration curves for the plasmid cDNAs
VEGF121,
VEGF165,
VEGF189, and for B2M cDNA (each
diluted 10-fold from 4 x 106 to 4 x
102 copies of plasmid cDNA per reaction mixture)
showed linearity over the entire quantification range with correlation
coefficients >0.99, indicating a precise log-linear relationship.
Because the amplicons of the three VEGF splice variants and
B2M have different sizes (range, 114–310 bp), PCR
efficiency may vary. However, for all dilutions of the four different
plasmid calibrators, the mean SD of the Ct values for each dilution
step was 0.21 cycles. This deviation was not significantly different
from the SD for the dilution series for the individual plasmid
calibrators (SD = 0.18 cycles). The slopes for the dilution series
for all four plasmid were 3.34–3.48, indicating comparable PCR
amplification efficiencies. Thus, for the different targets, all
experiments were performed using one calibration curve for all targets,
including B2M (Fig. 3
).
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reproducibility
The intrarun variability, calculated from triplicate samples for
all of the targets, showed an average SD for the Ct of 0.16 cycles. The
difference in absolute Ct values for each set of triplicates was never
>0.53 cycles. The interrun variability was assessed by comparing the
results of 14 different runs (corresponding to 14 different tissues)
using duplicates of the calibrator dilutions. When we compared the
means of two replicate measurements at each dilution, the difference in
the Ct was always <1.1 cycle, with a mean SD for all runs of 0.21
cycles. The slopes of the calibration curves of all 14 runs were
3.32–3.52.
vegf splice variants and
vegftotal expression in healthy and
malignant tissues
The measured copy numbers for
VEGFtotal cDNA were, on average, 90%
(SD = 5.6%) of the sum of the different VEGF splice
variant measurements in the 14 cDNA samples investigated. The
LightCycler PCR products of all 14 cDNA samples were collected, and gel
electrophoresis was performed. The copy numbers for the VEGF
splice variants in all 14 tissues are presented in Table 1
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Common immunological assays allow quantification of VEGF but fail to discriminate among isoforms because of a lack of VEGF isoform-specific monoclonal antibodies. Therefore, at present, investigation of the impact of differential expression of the various VEGF isoforms is possible only on the mRNA level. Several publications have indicated that expression of VEGF is controlled at the mRNA level by regulation of transcription rate and/or by changing mRNA stability (32)(33). To date, competitive RT-PCR has been the only method for semiquantitative detection of variant mRNA transcripts. This approach has two major disadvantages: (a) PCR endpoint analysis permits only semiquantitative analysis at a low sensitivity; and (b) formation of heteroduplexes between the different variants (34)(35), especially for the extensively spliced VEGF variants (36), further impairs quantification. In contrast, real-time quantitative RT-PCR, where quantification is performed during the exponential phase of amplification, permits accurate quantification over a wide dynamic range without any post-PCR processing. Heteroduplex formation does not play any role in real-time RT-PCR amplification. In the assay described here, each splice variant was separately and specifically amplified.
As shown in Fig. 4
, expression of the different VEGF splice variants is
detectable when the external primer pair for
VEGFtotal is used, but absolute
quantification of each variant as well as
VEGFtotal from the gel is not exact.
Therefore, statements concerning the absolute quantities of different
splice variants obtained by competitive PCR should be judged
critically.
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The presented standardized VEGF real-time RT-PCR assay using variant-specific reverse primers in combination with a common hydrolyzation fluorescent probe and a common forward primer ensures highly specific quantification with very good sensitivity and enables comparison of results generated by different investigators.
In this study, the mRNA message of VEGF splice variants and
VEGFtotal in 14 (7 healthy and 7 malignant)
tissues of different origin was investigated. The total VEGF
message was generally up-regulated in malignant tissues (20.5–728
copies/103 copies of B2M) compared
with healthy tissues (4.2–81 copies/103 copies
of B2M). Most remarkably, the copy number for
VEGFtotal and the sum of the different
VEGF splice variant copies showed excellent correlation (on
average, 90%; SD = 5.6%). In all 14 tissues, the splice variants
VEGF165 and
VEGF121 showed higher expression than
VEGF189. In healthy tissues, the mRNA
encoding diffusible variants VEGF121 and
VEGF165 constituted on average 78% (SD =
9.3%) of the total VEGF message, and the cell-adherent
variant VEGF189 constituted on average 22%
(SD = 5.4%). In contrast, in malignant tissues,
VEGF121 and VEGF165
constituted 94% (SD = 7.6%) of the total VEGF
message, whereas VEGF189 was 6% (SD =
3.7%). When we compared the absolute copy numbers for
VEGF189 in all healthy and malignant
tissues, no difference was found (Table 1
), whereas
VEGF165 and
VEGF121 were up-regulated in malignant
tissues. Our data, indicating that overexpression of VEGF in
tumors is caused by the more diffusible variants, support the
observations that during malignant progression an angiogenic switch
favoring the shorter diffusible isoforms (VEGF121
and VEGF165) occurs (27).
Because of the small number of correlated tissues investigated, differences in VEGF expression mark only a trend and allow no final conclusions. To verify the findings and to confirm the utility of the presented method, fresh malignant and healthy tissues from the same individuals should be measured. Furthermore, the prognostic significance of this assay could be demonstrated by examining malignant tissues from individuals with carcinomas in situ and by comparing with individuals with metastatic cancers.
In conclusion, the presented quantitative real-time RT-PCR assay clearly demonstrates many advantages over other existing methods for (semi)quantitative analysis of VEGF splice variants, including specificity, sensitivity, reduced carryover contamination, and rapid, accurate, and simultaneous quantification of multiple samples. Thus, we believe that this assay may be a promising tool for further investigations in the field of tumor angiogenesis and prognostic impact of VEGF in cancer.
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Acknowledgments |
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Footnotes |
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References |
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