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Nucleic Acids Research, 2003, Vol. 31, No. 1
DOI: 10.1093/nar/gkg011
#
2003 Oxford University Press
RTPrimerDB: the Real-Time PCR primer and
probe database
Filip Pattyn, Frank Speleman, Anne De Paepe and Jo Vandesompele*
Centre for Medical Genetics, Gent University Hospital, 1K5, De Pintelaan 185, B-9000 Gent, Belgium
Received August 14, 2002; Accepted September 1, 2002
ABSTRACT
The real-time polymerase chain reaction (PCR)
methodology has become increasingly popular for
nucleic acids detection and/or quantification. As
primer/probe design and experimental evaluation is
time-consuming, we developed a public database
application for the storage and retrieval of validated
real-time PCR primer and probe sequence records.
The integrity and accuracy of the data are maintained
by linking to and querying other reference databases.
RTPrimerDB provides free public access through the
Web to perform queries and submit user based
information. Primer/probe records can be searched
for by official gene symbol, nucleotide sequence,
type of application, detection chemistry, LocusLink
or Single Nucleotide Polymorphism (SNP) identifier,
and submitter’s name. Each record is directly linked
to LocusLink, dbSNP and/or PubMed to retrieve
additional information on the gene/SNP for which
the primers/probes are designed. Currently, the
database contains primer/probe records for human,
mouse, rat, fruit fly and zebrafish, and all current
detection chemistries such as intercalating dyes
(SYBR Green I), hydrolysis probes (Taqman), adjacent hybridizations probes and molecular beacons.
Real-time PCR primer/probe records are available at
http://www.realtimeprimerdatabase.ht.st.
INTRODUCTION
Since 1993, when the first polymerase chain reaction (PCR)
based DNA amplifications were on-line monitored (1), the
real-time PCR technology has successfully evolved to a
reliable and easy to use standard method for detection and/or
quantification of nucleic acid sequences. PCR product
accumulation is measured by a variety of fluorescent detection
chemistries, such as generic binding dyes, or sequence specific
probes like Taqman (including Minor Groove Binder probes)
(2), adjacent hybridization probes (3), or molecular beacons
(4). The reliability and accuracy of this method lies in part on
the specificity and reaction conditions of the primers and
probe(s). While there exists a large set of software tools and
Internet sites to design real-time primers and probes, timeconsuming and costly optimization experiments are required to
ensure that the designed oligonucleotides perform well.
Especially when working with highly homologous sequences
(e.g. gene families), it might prove difficult to design specific
working primers and probes for a particular transcript. We
noticed that many researchers experience the same problems
when designing real-time PCR primers and probes.
Furthermore, the parallel design of different primers for the
same sequence in different laboratories is often a waste of
resources, and excludes a certain level of standardization and
uniformity. As no specific database for this kind of information
is available, we initiated the development of a repository to
catalogue validated primer and probe sequences for real-time
PCR.
DETAILED INFORMATION
Implementation
RTPrimerDB is implemented on an Oracle 8i relational
database management system. The Web application to query
and manage the database is based on PHP scripts using the
Oracle Call Interface (OCI). The complete application runs on
an Apache web server in a Windows 2000 environment.
Data submission
We encourage real-time PCR users to submit their own,
validated, primer and probe oligonucleotides. Electronic data
submissions are possible after free registration. During
registration, personal submitter details are provided, after
which an email is sent with the registrar’s login name and a
temporary password. By changing this password to a more
convenient one, the registration is complete and the user can
log in to the system and submit custom primer sets. For
submission of large datasets, please contact us at administrator@realtimeprimerdatabase.ht.st.
New primer/probe records should contain the official gene
name or Single Nucleotide Polymorphism (SNP) identifier (5),
the species name and the application for which the primers are
used, the detection chemistry, the nucleotide sequences of
the primers and the probe(s), and the annealing temperature of
the primer pairs. Furthermore, each record provides the
*To whom correspondence should be addressed. Tel: þ32 9 2405518; Fax: þ32 9 2404970; Email: joke.vandesompele@rug.ac.be
Nucleic Acids Research, 2003, Vol. 31, No. 1
possibility to add submitter’s remarks. Currently, only data
submissions for primers/probes for human, fruit fly, mouse, rat
or zebrafish are allowed, as for these organisms proper controls
with respect to the accuracy of the gene name fields are available
via LocusLink (6) and the nomenclature databases for these
organisms: Genew (Human Gene Nomenclature Database) (7)
and GDB (8) for human, MGD (Mouse Genome Database) (9)
for mouse, Ratmap (10) and RGD (Rat Genome Database)
(11) for rat, FlyBase (12) for fruit fly and ZFIN (Zebrafish
Information Network) (13) for zebrafish. Doing so, the presence
of aliases or synonyms for official gene names is eliminated in
the database. Finally, the possibility to link the PubMed ID of an
article where the use of a primer set is discussed makes the
primer/probe data more trustworthy.
Querying for primers and probes
The on-line search engine makes it possible to query the
database in different ways by type of application, organism,
official gene name, detection chemistry, primer or probe
sequence, LocusLink ID, SNP identifier, or submitter’s name.
Search results are listed as a summary of links to the details
about specific primer/probe records, or as a complete list of all
the details. Each primer and probe set has a unique
RTPrimerDB identifier to access them directly or refer to in
a publication.
Data integrity
To guarantee accurate data, the sequences in the database are
analyzed on regular intervals by BLAST search against
the RefSeq database records (6). Upon detection of possible
sequence errors, the responsible submitter is contacted by
email.
Confidentiality and security
To protect the database information and submitters’ passwords,
we choose an Oracle database management system to manage
the access rights to the different tables and the submitters’
passwords.
FUTURE PLANS
In the coming months, we will migrate the data to an Oracle 9i
database management system and update the web server with
Secure Socket Layer (SSL) support. This will provide
improved security when login names and passwords are
transmitted. As the system allows easy inclusion of additional
applications, sequenced organisms, or newly developed
detection chemistries, we will extend the database with the
aforementioned items as soon as these become available or if a
reference database exists to guarantee the data integrity of the
database. In addition, the application will be accessible
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via a new, permanent URL. The current redirect address
http://www.realtimeprimerdatabase.ht.st will continue to operate to provide a link to the new URL.
ACKNOWLEDGEMENTS
We are grateful to Guy Detré and Axel Hallez of the Computer
Science Laboratory from the Faculty of Engineering of Ghent
University for their technical support during the development
of this database. Filip Pattyn is an Aspirant of the Fund for
Scientific Research of Flanders. This study was supported by
FWO-grant G.0028.00, GOA-grant 12051397 and BOF-grants
011B4300 and 011F1200.
There is a link to a FAQ section on the RTPrimerDB website
where further documentation is available on the use of the
database. For additional support or information regarding the
database, please write to help@realtimeprimerdatabase.ht.st.
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