TransPrimer: A Web-Based Tool for Automated Transcript-Specific RT-PCR/qPCR Primer Design Across Multiple Species

Transcript-specific RT-PCR/qPCR primer design is challenging when closely related RNA variants share extensive exonic sequence and differ primarily in exon connectivity. We developed TransPrimer, a web application for automated transcript-specific and pan-transcript primer design. TransPrimer retrieves curated RefSeq protein-coding and non-coding transcripts for six species and compares sibling transcripts to identify discriminating exons and exon-exon junctions (EEJs). The application automatically determines whether transcript specificity can be achieved using exon-targeted primers or requires single- or dual-EEJ strategies. A complementary pan-transcript mode identifies primer pairs that amplify all annotated transcripts of a gene, when feasible, or the largest compatible subset. Junction-spanning primers are evaluated using an arm-specific thermodynamic framework that independently calculates melting temperatures (Tm) for the full primer and its two exon-matching arms using nearest-neighbor thermodynamics and mixed-salt correction. Primer3-based criteria are integrated for partner-primer design and evaluation. User-adjustable reaction parameters, interactive transcript visualization, and custom sequence input support flexible assay development, including novel or externally annotated transcripts. By integrating transcript retrieval, structural comparison, strategy selection, thermodynamic evaluation, and primer generation, TransPrimer reduces manual transcript inspection and facilitates transcript-specific and gene-level RT-PCR/qPCR assay development across diverse transcript architectures.URL: https://transprimer.vercel.app/.