Experimentally Defined Tm-Guided Exon Exon Junction Primer Design Enables Specific Discrimination of Highly Similar RNA Variants

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 degree celsius below the annealing temperature, preventing stable binding to individual exons and enforcing junction dependent amplification. The method was evaluated using HTRA1AS1 long noncoding RNA variants that share overlapping exon sequences but differ in splice connectivity. HTRA1AS1 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.