Identification of growth-related tonoplast proteins in Arabidopsis thaliana

Background: Mediumto large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays. Results: Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in smallto large-scale qPCR analyses. QuantPrime can be used online through the internet http://www.quantprime. de/ or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%. Conclusion: QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broadrange platform for the design of RNA expression assays. Background The use of real-time quantitative PCR (qPCR) [1] in medium – (hundreds of transcripts) to large-scale (thousands of transcripts) profiling experiments is growing. While in a large number of experiments qPCR is still mainly used to confirm results obtained by microarraybased hybridization experiments, the number of highthroughput discovery experiments is growing steadily Published: 1 November 2008 BMC Bioinformatics 2008, 9:465 doi:10.1186/1471-2105-9-465 Received: 4 August 2008 Accepted: 1 November 2008 This article is available from: http://www.biomedcentral. com/1471-2105/9/465 © 2008 Arvidsson 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. BMC Bioinformatics 2008, 9:465 http://www.biomedcentral. com/1471-2105/9/465 [2,3], especially for the quantification of transcripts of low abundance (e.g. those coding for transcription factors), due to the low detection limit of the method [4]. There are surprisingly few free software packages available to the academic research community suitable for the design of primer pairs for such high-throughput projects, for online use or download, including Osprey [5], Primique [6] and a few interfaces to Primer3 [7] such as Primer3Plus [8], AutoPrime [9], BatchPrimer3 [10]. Additionally, some databases of pre-computed primers, RTPrimerDB [11], PrimerBank [12], qPrimerDepot [13], AtRTPrimer [14] and DATFAP [15], have been established. There are numerous commercial and free software packages available for low-throughput design of primers, some of which are highly configurable and well suited for the design of primer pairs for qPCR. However, none of the available packages combines all the important features (strict parameters for primer design, strict specificity checking and targeted design to avoid problems with contaminating genomic DNA) into a simple pipeline. Instead, with currently available computational tools, users have to either manually move information (such as identifiers, transcript sequences, primer sequences and others) between software packages or perform some steps completely on their own, such as specificity checking using an alignment package like BLAST [16]. Such manual steps make researchers loose valuable time, increase the risk of mistakes (e.g. labeling and sequence errors), and force them to take important decisions based on their personal interpretation of complex problems regarding large amounts of data (such as BLAST alignment sets), which either require expert knowledge or introduce bias into the results. With respect to the available primer pair databases, they are usually of limited scope. Often, only few species are covered (human and mouse being clearly over-represented), few transcripts of the species are represented (especially in databases based on submitted or published primer pairs), or inappropriate primer design parameters for combined analysis were used, requiring time-consuming optimization of PCR amplification conditions. Here we developed QuantPrime, a program for design and specificity testing of primer pairs for qPCR, designed to meet the needs of the average or advanced user in lowto high-throughput transcript profiling experiments, while keeping the user interface very simple and yet providing important features missing in other available software packages and web services. Implementation QuantPrime includes a relational database for information storage, scripts containing the procedures to perform primer pair design and specificity testing, scripts for sequence installation and maintenance, scripts for command line user interface used in high-throughput design, and a web interface as the main user interface for lowto medium-throughput primer design. For academic users we currently offer web access to the public QuantPrime server (available at http://www.quantprime.de/) or, on demand, compiled scripts for local installation. Commercial users are requested to get in contact with the authors to develop a license agreement. The public QuantPrime server is currently set up with publicly available transcriptome and genome annotations from 295 different eukaryotic species. Table 1 gives examples of supported species with included features and references. The list can be easily extended according to user requests. User interface The web interface is designed for maximum simplicity and convenience for the user. Users have to register at the first time they visit the website. The registration step allows users to return at a later time to check the results of longer runs. Their gene lists and jobs are kept confidential, i.e. no information is relayed to other users. Furthermore, registration eases the even distribution of computing resources among users and it is the main mechanism to verify academic affiliation. An account with access to limited computing resources is available for testing purposes. The work flow starts with the generation of a 'Project' that is associated with the annotation of a species and a certain quantification protocol. The quantification protocol implies certain parameters for primer design and specificity testing; four standard protocols for typical situations are provided: 1. SYBR Green-based real-time qPCR (accept splice variant hits): typical parameters for real-time qPCR are used, such as 50–150 bp amplicon length, 60°C annealing temperature and strict primer criteria for G/ C content and melting temperature (Tm). The specificity testing will allow amplicons present in splice variants of the transcript (more details in the 'Work flow' section). 2. SYBR Green real-time qPCR (no splice variant hits): as 1, but no amplicons in splice variants of the transcript are allowed. 3. End-point semi-quantitative PCR (accept splice variant hits): similar to 1, except that longer amplicons are preferred (350–1500 bp) for easier in-gel quantification. Page 2 of 15 (page number not for citation purposes) BMC Bioinformatics 2008, 9:465 http://www.biomedcentral. com/1471-2105/9/465 4. End-point semi-quantitative PCR (no splice variant hits): as 3, but no amplicons in splice variants of the transcript are allowed. Users are allowed to change any parameter and create custom protocols; see Additional file 1 for a list of all possible parameters. Next, users should create a list of transcript identifiers in the project for which primer pair design is planned. This list can either be entered manually (using the identifiers of the chosen annotation), or can be created from a similarity-based search using BLAST and a starting query sequence. Additionally, for certain annotations, keywords describing the gene(s) can be used in a text search for identifiers. Once the list of identifiers is ready, users may proceed to 'Primer finding' (Figure 1), which when started will continue completely in the background; in the meantime users can continue to look at resulting primer pairs or add new transcripts to the list. Larger primer finding projects may take longer time to process, therefore users may close the web browser and return at a later time to check the status of their jobs. Successful primer pairs are displayed in the 'Results' page (Figure 2), where users can inspect primer pairs in detail (Tm, G/C content, positions within transcript sequence etc., see example in Figure 3) and do bulk export of the primer data (in delimited plain text format) for ordering or local storage. Users may return at a later time to access their data, as lists of transcripts and primer pairs are automatically saved into their corresponding projects. On the public server, projects are kept for at least a month after the latest update, and may then be deleted by the administrator for space limitation reasons. Thus, users are recommended to export primer data and store locally for reference purposes. Work flow QuantPrime employs a fully aut