Tm-guided exon-exon junction RT-PCR enables specific detection of RNA variants lacking easily distinguishable exonic regions.

Accurate detection of RNA splice variants is often hindered when transcripts lack large distinguishable exonic regions, making conventional PCR strategies challenging. We developed a simple melting temperature (Tm)-guided exon-exon junction (EEJ) RT-PCR method to enable variant-specific detection under these conditions. Uni-directional primers spanning exon-exon junctions were designed so that approximately each half anneals to adjacent exons. The Tm of each half-site was set >7°C below the annealing temperature, preventing stable binding to individual exons and enforcing junction-dependent amplification. The method was evaluated using HTRA1-AS1 long noncoding RNA variants that share overlapping exon sequences but differ in splice connectivity. HTRA1-AS1 comprises five variants, only one with a large distinguishable exon. Tm-guided EEJ primers robustly discriminated the remaining four variants. After optimization, amplification yielded sharp, single bands with minimal cross-reactivity. Compared with conventional designs, this approach reduced heteroduplex and heteroquadruplex formation, improving band clarity. Sanger sequencing confirmed junction specificity, and the method performed well in multiplex settings. Overall, Tm-guided EEJ RT-PCR is a cost-effective, high-resolution approach for detecting RNA variants lacking easily distinguishable exonic regions, readily compatible with standard RT-PCR and qPCR workflows.