{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:42Z","timestamp":1792790382258,"version":"4.5.0"},"posted":{"date-parts":[[2026,4,5]]},"group-title":"Molecular Biology","reference-count":33,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,5]],"date-time":"2026-04-05T00:00:00Z","timestamp":1775347200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"name":"National Eye Institute","award":["P30 EY001765"],"award-info":[{"award-number":["P30 EY001765"]}],"id":[{"id":"https:\/\/ror.org\/03wkg3b53","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Gilbert Family Foundation","id":[{"id":"https:\/\/ror.org\/01kf4yp82","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Guerrieri Family Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Accurate detection of individual RNA transcript variants is challenging when closely related transcripts share extensive exonic sequence and differ primarily in exon connectivity. Analysis of curated human RefSeq transcripts identified 9,546 protein-coding genes containing at least one high-similarity RNA variant, highlighting the need for transcript-discriminating assays beyond conventional exon-targeted PCR. We developed a melting-temperature (Tm)-guided exon-exon junction (EEJ) RT-PCR strategy in which the 5\u2019 and 3\u2019 exon-matching arms of a junction primer are evaluated independently. Systematic experiments showed that productive amplification was lost when a partially matched primer segment had a Tm approximately 13-16 \u00b0C below the PCR annealing temperature, supporting an approximately 15 \u00b0C arm-specific design criterion. Tm-guided EEJ primers, including designs generated with the automated TransPrimer tool, specifically amplified highly similar transcript variants of HTRA1-AS1, APEX1, and GAPDH, reduced heteroduplex formation, and produced single qPCR melting peaks; most primer pairs showed 90-110% efficiency with R\n                  <jats:sup>2<\/jats:sup>\n                  &gt; 0.98. This approach provides an experimentally grounded framework for transcript-specific RT-PCR and qPCR when distinctive exonic regions are limited.\n                <\/jats:p>","DOI":"10.64898\/2026.04.02.716213","type":"posted-content","created":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T05:25:14Z","timestamp":1775453114000},"source":"Crossref","is-referenced-by-count":0,"title":["Experimentally Defined Tm-Guided Exon\u2013Exon Junction Primer Design Enables Specific Discrimination of Highly Similar RNA Variants"],"prefix":"10.64898","author":[{"given":"Junyeop","family":"Ahn","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, Stem Cell Ocular Regenerative Medicine Center, Wilmer Eye Institute, Johns Hopkins University School of Medicine; Baltimore, Maryland, 21231, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yun H.","family":"Lee","sequence":"additional","affiliation":[{"name":"Vugle Health LLC, Casper, Wyoming, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donald J","family":"Zack","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Stem Cell Ocular Regenerative Medicine Center, Wilmer Eye Institute, Johns Hopkins University School of Medicine; Baltimore, Maryland, 21231, United States"},{"name":"Solomon H. Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, Department of Genetic Medicine, Center for Nanomedicine at the Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping-Wu","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Stem Cell Ocular Regenerative Medicine Center, Wilmer Eye Institute, Johns Hopkins University School of Medicine; Baltimore, Maryland, 21231, United 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