QuantPrime--a flexible tool for reliable high-throughput primer design for quantitative PCR

Abstract

Background: Medium- to 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 small- to 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 broad-range platform for the design of RNA expression assays.

Figure 1

'Primer finding' in QuantPrime. The figure shows an example of the QuantPrime user interface for primer finding (A: up to nine transcripts, B: ten or more transcripts). The progress and success of the finding can be followed closely for small number of transcripts, for larger batches the time estimation helps users to estimate when the primer pairs will be ready.

Figure 2

'Results' in QuantPrime. The figure shows an example of the 'Results' page. Primer pairs successfully identified for the examined transcripts are presented. The following information is provided: the sequences (5' to 3') of the forward and reverse primers; the amplicon size (in bp); whether at least one primer spans an exon-exon junction ('Yes' in all cases in the example shown); the rank score (as calculated by Primer3); and the color code of the specificity rank given to the primer pair (see text for details). When clicking the primer pairs, more details are shown (see Figure 3).

Figure 3

Primer pair details in QuantPrime. The figure shows an example of the 'Primer pair information' page. The page provides details about the selected primers and the amplicon. Positions to which the primers anneal within the target sequence are indicated in blue or green; the amplicon is highlighted by gray shadowing. Primers shown in blue anneal to an exon, whereas primers shown in green anneal across an exon-exon junction (the position of the intron is indicated by a red arrow head). In the 'Specificity test results' section, details about the specificity of the primer pair can be seen. If specificity problems exist, more details can be found here concerning the other possible amplicons.

Figure 4

Overall work flow of primer pair design and specificity testing. Filled arrows symbolize logical flow while open arrows symbolize data flow.

Figure 5

Work flow overview of the primer pair design algorithm.

Figure 6

Work flow overview of the primer pair specificity testing algorithm.