Abstract
BackgroundPrimer design is a crucial step in establishing specific and sensitive qPCR assays. Even though numerous tools for primer design exist, the majority of resulting assays still requires extensive testing and optimisation or does not allow for high quality target amplification. We developed a workflow for designing qPCR assays. Unlike other tools, we compute a PCR assay including primer design, concentrations and the optimal PCR program.ResultsGene expression assays were already generated in a total of 283,226 genes from three species and are continued for all genes of the major model species. The results are available online at https://pcrdrive.com/lab#/assay-database. The workflow involves filtering Primer3-generated primers by considering diverse parameters including specificity, single-nucleotide polymorphisms (SNPs), secondary structure as well as compatibility with standard qPCR assay conditions. The resulting assays consist of transcript-specific primer sequences, a reagents protocol as well as instrument settings which are provided in a web-based tool called PCRdrive. PCRdrive was designed to support PCR users in their PCR-related tasks and is equipped with handy functions, components of an electronic lab notebook (ELN) as well as teamworking opportunities.ConclusionHigh quality ready to use qPCR assays for gene expression analysis are provided within the online platform PCRdrive. A built-in primer designer enables easy generation of assays which is not supported by any other tool. The wet lab optimisation of new assays can be transparently documented and shared within the team. PCRdrive also contains an archive of public PCRs which is updated regularly. Users may use the archive to publish their PCR to the community which makes it easy for other researchers worldwide to reproduce and validate the PCR. PCRdrive is a growing network of PCR users, simplifying and streamlining research through its useful existing features and continuous developments from the active development team.
Highlights
Primer design is a crucial step in establishing specific and sensitive Quantitative polymerase chain reaction (qPCR) assays
We show the number of considered input targets NT and the total number of generated Polymerase chain reaction (PCR) assays NA, which comprises all found gene variant categories
We have visualized the impact of the two main quality restrictions, Single nucleotide polymorphism (SNP) and cross-hybridisations, in Fig. 2 where we show the amount of primer pairs that needed to be discarded due to either of the two
Summary
PCR assay results Assay statistics For our sets of PCR assays we have gathered statistics related to the number of primer pairs that:. We show the number of considered input targets NT and the total number of generated PCR assays NA, which comprises all found gene variant categories. For Homo sapiens, for which the SNP database is even larger than for Mus musculus, finding valid primer pairs becomes even more difficult as can be seen from the left most bar of Fig. 2. The platform makes it easy for users to share his PCR work with others (be it a copied and adapted qPCR assay or a primer pair generated by the built-in primer designer), for which several opportunities exist. PCRdrive allows one to select a team administrator and post or edit PCRs jointly This private, collaborative space is an easy way to share each step of the process and trade information in a secure and organized group. It is possible to share a PCR directly with any other PCRdrive user using an inbox/outbox like mechanism known from regular email communication
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