Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas.

Abstract

Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator SMART cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis.
Random analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found.
Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.

Full text

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BioMed Central
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Genetic Vaccines and Therapy
Open Access
Research
Production and characterization of amplified tumor-derived cRNA
libraries to be used as vaccines against metastatic melanomas
Jean-Philippe Carralot1,2, Benjamin Weide3, Oliver Schoor2, Jochen Probst1,2,
Birgit Scheel1, Regina Teufel1, Ingmar Hoerr1, Claus Garbe3, Hans-
Georg Rammensee2 and Steve Pascolo*1,2
Address: 1CureVac, Paul Ehrlich Strasse 15, 72076 Tübingen, Germany, 2University of Tübingen, Institute for Cell Biology, Department of
Immunology; Auf der Morgenstelle 15; 72076 Tübingen, Germany and 3Section for Dermatological Oncology, Tübingen University Hospital,
Liebermeisterstraße 25, 72076 Tübingen, Germany
Email: Jean-Philippe Carralot - carralot@curevac.de; Benjamin Weide - bnweide@med.uni-tuebingen.de; Oliver Schoor - oliver.schoor@uni-
tuebingen.de; Jochen Probst - jochen.probst@student.uni-tuebingen.de; Birgit Scheel - bs@curevac.de; Regina Teufel - rt@curevac.de;
Ingmar Hoerr - ih@curevac.de; Claus Garbe - claus.garbe@med.uni-tuebingen.de; Hans-Georg Rammensee - rammensee@uni-tuebingen.de;
Steve Pascolo* - steve.pascolo@uni-tuebingen.de
* Corresponding author
Abstract
Background: Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an
attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated
metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the
large quantities of mRNA needed for each patient, the Stratagene Creator™ SMART™ cDNA
library construction method was modified and applied to produce libraries derived from the
tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through
sequencing and microarray analysis.
Results: Random analysis of bacterial clones of the library showed a rate of 95% of recombinant
plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In
addition, despite a biased amplification toward small abundant transcripts compared to large rare
fragments, we could document a relatively conserved gene expression profile between the total
RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA
(cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number
of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were
found.
Conclusion: Our results show that unlimited amounts of cRNA representing tumor's
transcriptome could be obtained and that this cRNA was a reliable source of a large variety of
tumor antigens.
Published: 22 August 2005
Genetic Vaccines and Therapy 2005, 3:6doi:10.1186/1479-0556-3-6
Received: 22 June 2005
Accepted: 22 August 2005
This article is available from: http://www.gvt-journal.com/content/3/1/6
© 2005 Carralot et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
The identification by van der Bruggen et al. [1] of the first
tumor associated (TAA) antigen recognized by specific
cytotoxic T lymphocytes (CTLs) in melanoma patients
boosted the development of anti-cancer immunotherapy
strategies. During the last years, vaccination protocols tar-
geting differentiation antigens (MART-1/Melan-A [2,3],
gp100 [4], Tyrosinase [5,6]) or cancer-testis antigens
(MAGE [1,7], NY-ESO1 [8]) were tested and showed
encouraging results [9-11].
However, a growing body of evidence suggests that,
instead of using defined antigens, targeting the whole
spectrum of tumor antigens would represent an alterna-
tive, potentially more efficacious method [12-14]. Indeed,
the use of total tumor material for vaccination allows the
development of B and T cells directed against a large vari-
ety of known but also unknown TAAs [15]. In addition,
stimulating such a large spectrum of specific effectors
directed against multiple epitopes restricted by diverse
HLA class I and II types would reduce the risk of tumor
escape through antigen loss or MHC downregulation [16-
19]. Finally, another advantage of the whole tumor
approach is that, in an autologous setting, patient's TAAs
eventually stemming from tumor-specific somatic muta-
tions could be targeted [20,21].
In order to vaccinate patients with the whole spectrum of
TAAs, several methods were developed. In 1998, Soiffer et
al. [22] disclosed the results obtained by vaccinating
patients with autologous irradiated tumor cells engi-
neered to produce GM-CSF. The same year, Nestle et al.
[23] showed partial or complete tumor remissions in six
melanoma patients vaccinated with dendritic cells (DC)
loaded with autologous tumor lysate. Alternatively, Bocz-
kowski et al. [24] reported that mouse DCs pulsed in vitro
with tumor RNA could trigger an anti-tumor immunity in
vivo. Several groups further developed and optimized
those different strategies [25-27] but faced the limitation
imposed by the requirement of large amounts of tumor
tissue for lysate preparation or for sufficient RNA yields
extraction. In order to overcome this drawback, Bocz-
kowski et al. [28] modified the SMART method (BD Bio-
sciences Clontech, Palo Alto, CA) in order to in vitro
transcribe tumor cDNA and performed therefore a one-
step amplification of tumor mRNA. Transfected into anti-
gen presenting cells (APCs), this amplified cRNA was
shown in vitro to induce anti tumor immunity [29,30]. As
an alternative vaccination method, Hoerr et al. [31] dem-
onstrated the capacity of mRNA coding for defined anti-
gens or of total cRNA to trigger an antigen-specific
immune response after direct intra-dermal injections of
the ribonucleic acid. Similarly, Granstein et al. [15]
showed protection against S1509 tumor cells in mice that
received three intradermal injections of total RNA
extracted from S1509 cells. Although still marginally stud-
ied compared to mRNA-loaded DC vaccines, the direct
injection of mRNA represents a technology that offers the
important advantage to circumvent the time and money
consuming steps of generation of DCs.
In 2003, we initiated the first phase I/II clinical study to
test the feasibility, safety, and efficacy of a vaccine com-
posed of autologous amplified tumor mRNA in stage III/
IV patients with metastatic melanoma (The detailed eval-
uation of the toxicity, clinical and immunological efficacy
of this treatment will be reported in a following manu-
script). Fifteen patients received from 3 to 16 intradermal
injections of 200 µg of amplified autologous tumor cRNA.
The amount of injected RNA was limited by the maximal
intradermal injection volume (100 µl) and set according
to the preclinical results which indicated that a concentra-
tion of ca. 0.8 µg/µl was leading to a good gene transfer.
The injection's schedule consisted in applications every
two weeks of four injections and then once every month.
It was decided empirically since no previous data on the
toxicity and efficacy of this immunization method are
available in humans. In mice one injection was shown to
be sufficient to trigger an immune response [31]. How-
ever, in cancer patients, a sustained stimulation of the
immunity is probably requested in order to get an efficient
anti-tumor immune response. According to this protocol,
the required amount of cRNA for a complete therapy was
between 0.6 and 3.2 mg per patient (table 1). In order to
get unlimited amounts of product, a new method for the
amplification of the tumor mRNA was developed. Briefly,
a cDNA library was generated from tumor RNA using the
SMART (Switch Mechanism At the 5'end of RNA Tem-
plates) system (BD Clontech) and then was cloned in our
RNActive™ vector (CureVac GmbH), amplified in
Escherichia coli, and finally transcribed in vitro. As opposed
to the protocol described by Boczkowski et al. [28] in
which the PCR-amplified tumor cDNA library was directly
used as template for the in vitro transcription (resulting in
limited cRNA amounts), the method applied in our labo-
ratory provided us with unlimited amounts of the tumor-
derived cRNA.
Whereas the SMART method was reported to maintain the
relative levels of RNAs contained in the original transcrip-
tome regardless of their size or their baseline expression
[32], the cloning step in E. coli was on the contrary
described to introduce a bias favoring short fragments
[33]. We thus analyzed the quality of the produced ampli-
fied-mRNA libraries to be used as a vaccine in melanoma
patients. Several clones randomly picked-up within the
produced libraries were analyzed by PCR and sequenced.
In addition, the gene expression profiles of two metastases
were compared to their corresponding cRNA-libraries.

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Results and discussion
Tumor-derived mRNA library quality
Total RNA was extracted form 15 fresh melanoma tissues.
It was then used to generate cDNA libraries according to
the SMART protocol (BD Clontech, Palo Alto, CA). The
obtained cDNA libraries were ligated into the RNActive™
vector (CureVac GmbH, Tübingen, Germany) containing
the mRNA stabilizing sequences of 5' and 3' UnTranslated
Regions (UTR) from β- and α-globin respectively [34,35].
Moreover, this vector introduced a 70 A poly(A) tail fur-
ther enhancing mRNA translation potential (data not
shown). RNActive™ libraries were transformed into ultra-
competent E. coli. The total primary transformant num-
bers were ranging from 6 × 104 to 5 × 105 clones with an
average number of 2 × 105 (table 1). In order to determine
the ligation efficiency, 24 clones per library were ran-
domly picked up and submitted to 35 PCR cycles using
primers flanking the cDNA insertion sites. Ninety-eight
percent of the 312 analyzed clones had an insert with sizes
ranging from 200 bp to 10 kbp (data not shown). In order
to further test the quality of the libraries, plasmid DNA of
9 clones randomly picked up were extracted for 5'end
sequencing (Figure 1). Out of the 112 readable sequences,
2 clones had no insert confirming the 2% rate of self-liga-
tion found by PCR analysis. Among the remaining 110
clones, 3 (3%) showed sequences classified as aberrant
with insert sizes inferior to 50 bp probably corresponding
to recombination events. The other 107 sequences were
analyzed BLAST [36] (Basic Local Alignment Search Tool)
using the nr database. About half of the clones (51%) cor-
responded to full Open Reading Frames (ORFs) of anno-
tated sequences. The other sequences were homologous
to ESTs (Expressed Sequence Tags) coding for potential
proteins with unknown functions. Full-length clone sizes
ranged from 344 to 5925 bp with an average size of 1395
bp correlating with the average insert size of 1.4 kb in
cDNA libraries described by Draper et al. [37]. Interest-
ingly, 28 % of the clones which aligned to annotated
ORFs (14% of all sequenced clones) were tumor related
genes (for instance S100 Calcium binding protein A4-
metastasin [38]), or genes reported to be overexpressed in
tumors (for instance Laminin receptor [39]). This obser-
vation fitted with the objective of using the cRNA libraries
as anti-tumor vaccines.
Relative representation of transcripts
In order to determine whether a bias was introduced by
the amplification protocol, the relative gene expression in
extracted total RNA from two metastases was compared to
the relative gene expression in the corresponding ampli-
fied cRNA libraries. Biotin-labeled complementary RNA
of tumor total RNA and of amplified cRNA libraries from
patients MEL02 and MEL10 were generated using the
Affymetrix eukaryotic sample and array processing stand-
ard procedure and hybridized on HG-U133A microarrays.
According to the Microarray Analysis Suite 5.0 software
(MAS 5.0; Affymetrix), 34% and 36% of the genes that
were reported as "present" in the tumor total RNA were
also detected as "present" in the amplified libraries of
patients MEL02 and MEL10 respectively (Theses
Table 1: Summary of mRNA libraries and clone analysis. In the case of MEL14, total RNA was extracted from ~5 × 104 pleural tumor
cells (NA: Not applicable)
PatientsWeight of
tumor sample
(mg)
Quantity of
extracted
total RNA
(µg)
Number of
clones (cfu)
Size range of
analyzed
clones (nt)
Quantity of
mRNA library
prepared
(mg)
Number of
injection
performed
1
2
3
4
5
6
7
8
9
MEL01
MEL02
MEL03
MEL04
MEL05
MEL06
MEL07
MEL08
MEL09
MEL10
MEL11
MEL12
MEL13
MEL14
MEL15
32.4
33
33.6
38
85
60
76
34.1
95
78.7
77.3
34.3
72
NA
60
9.4
10.5
26.3
36.7
14.2
115.2
70.5
60.9
84
62.5
9.56
15.4
9.14
13.2
41.5
1 × 105
1 × 105
5 × 105
2 × 105
2 × 105
3 × 105
5 × 105
3 × 105
4 × 104
2 × 105
3 × 105
6 × 104
2 × 105
1 × 105
3 × 105
500 – 4000
200 – 8000
250 – 1000
400 – 3500
500 – 1000
300 – 1200
500 – 1200
500 – 1200
350 – 800
600 – 1200
400 – 1000
400 – 1200
750 – 2000
400 – 10000
500 – 4000
2.8
5.0
1.9
3.6
5.0
4.0
2.8
5.3
4.5
4.2
2.7
3.9
4.4
1.8
4.1
10
12
6
8
13
16
7
16
10
16
3
10
16
4
8
10
11
12
13
14
15
Average
57.838.6 2 × 105
450 – 32503.7 10

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transcripts are qualified as "recovered" in the following).
The other transcripts reported by the Microarray Analysis
Suite 5.0 software as "present" in tumor's mRNA but as
"absent" in the corresponding cRNA library were termed
as "lost". In order to determine the factors influencing the
biased amplification of genes, the size distributions of
"lost" and "recovered" transcripts were compared and
plotted in figure 2A. For both patients MEL02 and MEL10,
the average size distribution of "recovered" genes (2218
and 1965 nt respectively) was significantly lower (t test, P
< 0.0001) than the average size of transcripts lost during
the amplification process (2894 and 3222 nt respec-
tively). This suggests a biased amplification disfavoring
large fragments as observed by Wellenreuther et al. [33].
In addition, the fluorescence values in the original tumor
of genes reported as "recovered" and "lost" in the cRNA
were compared as shown in figure 2B. Average signals of
2711 and 3709 for MEL02 and MEL10 respectively were
observed for the genes present in the cRNA library and
thus preserved during the process. In contrast, the genes
"lost" during amplification had an average signal of only
of 708 and 1174 for patients MEL02 and MEL10 respec-
tively. These values were significantly lower (t test, P <
0.0001) than those found for the group of "recovered"
genes. Thus, transcripts of higher abundance in the origi-
nal tumor were preferentially preserved during the ampli-
fication process whereas mRNAs of lower abundance were
eventually lost.
The fluorescence signal intensities of the genes reported as
"present" in both the tumor and the corresponding cRNA
libraries were plotted in figure 3. The correlation factors to
a linear regression were 0.55 and 0.42 for patients MEL02
and MEL10 libraries respectively, confirming that the
mRNA amplification was quite heterogeneous. However,
in the group of "recovered" transcripts no significant cor-
relation was evidenced between the transcript size or their
signal intensity in the tumor of origin and their amplifica-
tion factor during the cloning (data not shown).
Patient-specific gene expression
In order to evaluate the relevance of the autologous
approach, signal intensities for the genes present in metas-
tases of MEL02 and MEL10 were compared in figure 4. As
expected, the two melanoma samples were quite similar
with a correlation coefficient to a linear regression of 0.75
for the 6693 genes shared between the two tumors. How-
ever, the two tissues showed specific profiles with 3222
and 483 mRNA transcripts expressed only in patient's
MEL02 and MEL10 metastasis respectively. These patient-
BLAST analysis of sequenced clones
Figure 1
BLAST analysis of sequenced clones. Nine clones per library were randomly picked up and their plasmid DNA was
sequenced using a T7 promoter primer. Readable sequences (n = 112) were submitted to a BLAST analysis and their relative
distribution plotted.
Tumor-related
genes
14%
Other genes
37%
Empty clones
2%
Recombinant
3%
Genes with
unknown functions
44%
Genes withknown
functions
51%

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specific antigens might represent particularly interesting
immunological targets [13] and argue for the injection of
autologous tumor cRNA rather than the use of cRNA
library derived from tumor cell lines as a vaccine.
Several tumor antigens are present in the cRNA libraries
The transcripts showing the highest fluorescence signals
in the injected cRNA library were listed in Table 2 for
patient MEL02. Among the 30 genes displaying the high-
est signals likely representing most abundant transcripts,
13 have already been described as being involved in
tumor genesis or observed as being overexpressed in dif-
ferent cancer types. The well-defined and widely used
tumor antigen Melan-A was found as one of the most
abundant transcripts showing the relevance of cRNA
libraries as melanoma vaccines. In addition, the presence
in the injected library of many other "tumor-related" anti-
gens rarely used in vaccines targets highlights the poten-
tial of such a product to vaccinate patients against a large
panel of TAAs.
Comparison of sizes and fluorescence signal intensities in the original tumor sample for "lost" and "recovered" transcripts
Figure 2
Comparison of sizes and fluorescence signal intensities in the original tumor sample for "lost" and "recovered"
transcripts. A. The sizes of transcripts Present in tumor's total RNA and reported as Present (PP) or Absent (PA) in the
cRNA library of MEL02 and MEL10 patients were plotted. The average size of "recovered" genes was significantly lower (t test,
P < 0.0001) than the average size of "lost" genes. B. The fluorescence signals of genes "Present" in the original tumor were
compared for the transcripts reported as Present (PP) or Absent (PA) in the corresponding mRNA libraries. The group of
"recovered" genes showed a significantly higher (t test, P < 0.0001) mean signal than the group of genes "lost" during the library
production.
A
MEL02
2218
2894
100
1000
10000
100000
Transcript size (nt)
PP
(n=570)
PA
(n=1021)
MEL10
1965
3322
100
1000
10000
100000
Transcript size (nt)
PP
(n=545)
PA
(n=615)
B
MEL10
3709
1174
10
100
1000
10000
100000
Fluorescene signal
PP
(n=3259)
PA
(n=4161)
MEL02
2711
708
10
100
1000
10000
100000
Fluorescence signal
PP
(n=3676)
PA
(n=6607)

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Conclusion
In order to vaccinate metastatic melanoma patients with
autologous amplified tumor-derived cRNA, fifteen
libraries were produced using a modified SMART method.
Despite a heterogeneous amplification of tumor genes,
this method provided us with an unlimited source of
tumor and patient specific TAAs. Indeed, the microarray
analysis of the libraries indicated the presence of high
copy numbers of well-known tumor associated antigens
such as Melan-A but also of abundant tumor-related anti-
gens scarcely targeted in immunotherapy. Although not
addressed in the present work, this method might also
allow the targeting of tumor-specific mutations. These fea-
tures makes of the amplification of tumor mRNA the
method of choice to easily obtain unlimited amounts of
RNA coding for patient's specific TAAs that can be applied
as anti-tumor immunotherapy.
Materials and methods
Tumor samples
Immediately after surgery, metastatic tissues from fully
informed patients (Ethic committee approval Nr.: 269/
2002) were chopped in ~0,1 cm3 pieces, and submerged
in RNAlater solution from Ambion (Hungtingdon, UK),
and stored at 4°C until histological identification as
melanoma by an experienced pathologist.
Tumor total RNA extraction
Total RNA was extracted from tumors using the RNeasy
mini kit from Qiagen (Hilden, Germany) following the
instructions of the provider. Briefly, 15 to 30 mg samples
placed in a 2 ml eppendorf tube were snap-frozen in a liq-
uid nitrogen bath and disrupted with micropistils from
Eppendorf (Hamburg, Germany). Tumor powder was
resuspended in RLT buffer, homogenized through a 20-
gauge needle and digested with 200 µg of proteinase K
(Qiagen) at 55°C during 10 min. Samples were then clar-
ified, loaded on RNeasy mini columns, washed and
finally eluted in 50 µl of RNAse-free water. RNA was quan-
tified by U.V spectrophotometry (O.D260/O.D280 ratio was
over 1.8 in all cases) and analyzed on a 1,2% formalde-
hyde/agarose gel.
cDNA library generation
cDNA libraries of tumor total RNA were generated using
the slightly modified Creator™ SMART™ PCR cDNA
library construction kit from BD Biosciences Clontech
(Heidelberg, Germany). Briefly, 1 µg of total RNA was
reverse transcribed using SMART IV™ and CDS III/3' oligo-
Correlation of signal intensities in tumor and corresponding mRNA libraries for patients MEL02 and MEL10
Figure 3
Correlation of signal intensities in tumor and corresponding mRNA libraries for patients MEL02 and MEL10.
Fluorescence signals in original metastases and amplified tumor cRNA libraries for patients MEL02 and MEL10 were compared
for all genes reported as "Present" in the library by MAS 5.0 software.
MEL02
slope = 1.7011
R2 = 0.5532
0
20000
40000
60000
80000
100000
020000400006000080000 100000
Fluorescence signal in tumor
Fluorescence signal in library
MEL10
slope = 0.8021
R2 = 0.4187
0
20000
40000
60000
80000
100000
0200004000060000 80000 100000
Fluorescence signal in tumor
Fluorescence signal in library

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dT primers provided by the manufacturer. After termina-
tion of the reaction, 2 µl of cDNA were amplified using
the Advantage 2 PCR kit (BD Biosciences Clontech). DNA
polymerase was then inactivated with proteinase K and
the cDNA library was digested with 200 U of Sfi I enzyme.
cDNA libraries were then gel-purified on an 1% agarose
gel and fragments from 300 bp to 10 kbp were extracted
using E.Z.N.A.™ Gel Extraction kit from Peqlab GmbH
(Erlangen, Germany). After precipitation, the cDNA
library was ligated to dephosphorylated Sfi I-digested
RNactive™ vector provided by CureVac GmbH (Tübingen,
Germany) in three separated reactions to optimize vector/
insert ratios.
Cloning
The three ligation products were used to transform XL10-
Gold ultracompetent cells from Stratagene (Heidelberg,
Germany). For analysis, 1 and 10 µl of transformation
broth were plated on 2 LB-ampicillin agar plates and, after
overnight culture at 37°C, the number of clones was
counted. The inserts of 8 clones per transformation were
amplified by PCR using primers flanking the insertion
sites and amplicons were analyzed on a 1% agarose gel.
Libraries having more than 104 clones/ml and less than
20% of non recombinant clones, were amplified in three
300 ml maxicultures in 2X LB-ampicillin medium during
20 h at 33°C in order to limit uneven amplification of
clones.
DNA preparation and linearization
Maxicultures were pooled, centrifuged down at 5 000 rpm
for 10 min and plasmid DNA was extracted using
EndoFree Plasmid Maxi (Qiagen). After precipitation, 100
µg of cDNA library were digested with 100 U of Not I
enzyme. After phenol/chloroform extraction and ammo-
niumacetate precipitation, linearized cDNA libraries were
resuspended in RNAse-free water, quantified by U.V. spec-
trophotometry (O.D260/O.D280 ratio was over 1.8 in all
cases) and analyzed on 1% agarose gel.
cRNA In vitro transcription
Twenty to hundred micrograms of linear cDNA library
were in vitro transcribed using T7 mRNA Optikit from
CureVac GmbH. After mRNA synthesis, DNA template
was digested with 40 to 100 U of recombinant DNAse I
purchased from Ambion. mRNA was then LiCl precipi-
tated, phenol/chloroform purified, NaCl precipitated,
and finally resuspended in PBS. cRNA was filter sterilized
(0,2 µm), heat denatured at 80°C for 10 min before final
sterile aliquoting. cRNA was quantified by U.V
spectrophotometry (O.D260/O.D280 ratio was over 1.8 in
all cases) and analyzed on 1.2% formaldehyde/agarose
gel. Sterility of cRNA was checked by inoculating LB
medium (in all cases, no bacterial growth was observed
after 4 days at 37°C) and endotoxin content was deter-
mined using Bio-Whittaker (Verviers, Belgium) LAL assay
kit (endotoxin content was always below 7 EU/ml).
Clone sequencing
For each library, 3 colonies per transformation were ran-
domly picked-up with a pipette tip and used to inoculate
3 ml of LB-ampicillin medium. After overnight culture at
37°C, plasmid DNA was extracted using E.Z.N.A
miniprep kit (Peqlab). Clone sequencing was performed
using the ABI Big Dye and a T7 promoter primer.
Sequences were purified on Autoseq. G-50 columns
(Amersham Pharmacia Biotech, Freiburg, Germany), run
on a 310 Genetic Analyzer from ABI PRISM™ (Applied
Biosystems, Darmstadt, Germany) and analyzed with the
Sequencing Analyzing 3.4.1 software (ABI PRISM).
Finally, the BLAST algorithm [36] was used to identify
matches to known genes.
Microarray analysis
Expression analysis of total tumor RNA and amplified
tumor cRNA was performed on HG-U133A microarrays
from Affymetrix (High Wycombe, UK) according to the
manufacturer's eukaryotic sample and array processing
standard procedure [40], which is based on the IVT
method originally described by Van Gelder et al. [41].
Briefly, 1st-strand cDNA synthesis was performed using an
Correlation of fluorescence of genes present in tumors of patients MEL02 and MEL10
Figure 4
Correlation of fluorescence of genes present in
tumors of patients MEL02 and MEL10. Fluorescence
signals of patients MEL02 and MEL10 tumor transcripts
reported by MAS 5.0 software as present in both samples
(6993 genes, ◆), only in MEL02 metastasis (3222 genes, ●)
or only in MEL10 melanoma (483 genes, ▲).
slope = 1.3789
R2 = 0.7329
0
10000
20000
30000
40000
50000
0100002000030000 4000050000
MEL02
MEL10

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oligo(dT)24 primer containing a T7 promoter sequence.
After RNA template degradation and cDNA's second
strand cDNA synthesis, complementary RNA (cRNA) was
transcribed in vitro using biotinylated NTPs and T7 RNA
polymerase. After purification using RNeasy columns
(Qiagen), 18 µg of biotin-labeled cRNAs were fragmented
by metal-induced hydrolysis. Hybridization, staining, and
scanning of microarrays were performed by the Microar-
ray Facility Tübingen. Scanned images were processed
using the Microarray Analysis Suite 5.0 (MAS 5.0; Affyme-
trix) and expression differences between tumor and
library samples were determined by baseline comparison
algorithms provided by the software. Data were further
processed using Microsoft Access™ and Excel™.
Authors' contributions
JPC carried out the total tumor RNA extraction, the pro-
duction of mRNA libraries, the analysis of clones, partici-
pated in the microarray analysis of RNA samples,
evaluated the microarray data and drafted the manuscript.
OS carried out the microarray analysis of RNA samples
with the help of the Microarray Facility Tübingen. BW and
CG carried out the patient's recruitment and the tumor
sample preparation. JP, BS and RT participated in the
design of the study, the development of the work and
helped to draft the manuscript. CG, IH, HGR and SP con-
ceived, designed and coordinated this study. All authors
read and approved the final manuscript.
Acknowledgements
JPC is supported by a "Fortüne" grant from the University of Tübingen and
JP is supported by the DFG Graduiertenkollegue "Infektionsbiologie" of
Tübingen.
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Table 2: List of the thirty transcripts showing the highest fluorescence signals in MEL02 amplified cRNA library
TitleSymbolObservations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Ribosomal protein L23aRPL23a
EEF1A1
RPS3A
RNASE1
PPIA
RPS23
RPL39
MLANA
RPL31
COX6C
RPL7
RPL37A
RPS29
SPP1
CALM2
RPS11
RPS4X
NACA
RPL23A
ATP5L
K-ALPHA-1
NDUFA4
RPS27A
B2M
H2AFZ
SOX4
ATP5E
TPT1
COX7A2
UBB
Eukaryotic translation elongation factor 1 alpha 1
Ribosomal protein S3A
RNase A family, 1 (pancreatic)
Peptidylprolyl isomerase A, cyclophilin A
Ribosomal protein S23
Ribosomal protein L39
Melan-A
Ribosomal protein L31
Cytochrome c oxidase subunit VIc
Ribosomal protein L7
Ribosomal protein L37a
Ribosomal protein S29
Secreted phosphoprotein 1, osteopontin
Calmodulin 2
Ribosomal protein S11
"Ribosomal protein S4, X-linked"
Nascent-polypeptide-associated complex alpha
Ribosomal protein L23a
ATP synthase, mitochondrial F0 complex, subunit g
Tubulin, alpha, ubiquitous
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex
Ribosomal protein S27a
Beta-2-microglobulin
H2A histone family, member Z
SRY (sex determining region Y)-box 4
ATP synthase, mitochondrial F1 complex, epsilon subunit
Tumor protein, translationally-controlled 1
Cytochrome c oxidase subunit VIIa polypeptide 2
Ubiquitin B
Overexpressed in several cancers [42]
Melanoma differentiation antigen [2]
Overexpressed in carcinomas [43]
Overexpressed in gliomas [44]
Important for tumorgenesis [45]
Overexpressed in several cancers [42]
Overexpressed in gliomas [44]
Involved in tumor proliferation [46]
Overexpressed in breast cancers [47]
Overexpressed in breast cancers [48]
Overexpressed in several cancers [42]
Overexpressed in lung cancers [49]
Involved in malignant transformation [50]
Related to sustained proliferation [51]

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