STALARD: Selective Target Amplification for Low-Abundance RNA Detection

STALARD: Selective Target Amplification for Low-Abundance RNA Detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article STALARD: Selective Target Amplification for Low-Abundance RNA Detection Daesong Jeong, Ilha Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6782090/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Plant Methods → Version 1 posted 9 You are reading this latest preprint version Abstract Background Accurate quantification of transcript isoforms is critical for understanding gene regulation. However, conventional reverse transcription-quantitative real-time PCR (RT-qPCR) has limited sensitivity for low-abundance transcript isoforms, as quantification cycle (Cq) values above 30 are often considered unreliable. While transcriptome-wide analyses can address this limitation, they require costly deep sequencing and complex bioinformatics. Results To overcome the sensitivity limitations of conventional RT-qPCR for detecting low-abundance and alternatively spliced transcripts, we developed STALARD (Selective Target Amplification for Low-Abundance RNA Detection), a rapid (<2 hr) and targeted two-step RT-PCR method using standard laboratory reagents. STALARD selectively amplifies transcripts sharing a common 5′-end, enabling efficient quantification of low-abundance isoforms. When applied to Arabidopsis thaliana, STALARD successfully amplified the low-abundance VIN3 transcript to reliably quantifiable levels. Amplification of FLM, MAF2, EIN4, and ATX2 isoforms by STALARD reflected known splicing changes during vernalization, including cases where conventional RT-qPCR failed to detect relevant isoforms. STALARD also enabled consistent quantification of the extremely low-abundance antisense transcript COOLAIR, resolving inconsistencies reported in previous studies. In combination with nanopore sequencing, STALARD further revealed novel COOLAIR polyadenylation sites not captured by existing annotations. Conclusion STALARD provides a sensitive, simple, and accessible method for isoform-level quantification of low-abundance transcripts. Its compatibility with both qPCR and long-read sequencing makes it a versatile tool for analyzing transcript variants and identifying previously uncharacterized 3′-end structures. Low-abundance RNA Reverse transcription-quantitative real-time PCR Selective transcript enrichment Full Text Additional Declarations No competing interests reported. Supplementary Files STALARDSupplementaryFigures.docx STALARDSupplementaryTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Plant Methods → Version 1 posted Editorial decision: Revision requested 21 Jul, 2025 Reviews received at journal 05 Jul, 2025 Reviewers agreed at journal 02 Jul, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 09 Jun, 2025 Reviewers invited by journal 03 Jun, 2025 Editor assigned by journal 31 May, 2025 Submission checks completed at journal 31 May, 2025 First submitted to journal 30 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6782090","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":465945172,"identity":"3822355f-feb1-4c22-95cf-7fd95379c53f","order_by":0,"name":"Daesong Jeong","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Daesong","middleName":"","lastName":"Jeong","suffix":""},{"id":465945173,"identity":"76473e44-9585-4dd0-8ce1-87a9e63969fe","order_by":1,"name":"Ilha Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACNnYeBoYHDAxyMAEDwlqYgVoSGBiMidfCANWS2EC0Fj5m3oMfEips0ufP7jFg+FHDYGzeQEALGzNfskTCmbTcDXfOGDD2HGMwkzlAUAuPgURi2+HcDRI5Bgy8DQw2EoQcBtRi/CPx3/90+Rk5Box/idRiJpHYcCCB4UaOATPQFjOitFgkHEs23HAjreCwzDEJY4Ja5Nt7jG98qLGTl5+RvPHhmxobwxmEtKCAAwwMBO0YBaNgFIyCUUAMAADS8jPvZZyVlgAAAABJRU5ErkJggg==","orcid":"","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Ilha","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-05-30 07:23:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6782090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6782090/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13007-025-01443-z","type":"published","date":"2025-09-29T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92883822,"identity":"01858269-df54-449d-88e5-093ba6c61be0","added_by":"auto","created_at":"2025-10-06 16:10:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":556792,"visible":true,"origin":"","legend":"","description":"","filename":"STALARDmanuscriptwithfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6782090/v1_covered_313eac43-d27f-4956-adef-3bcf5139e387.pdf"},{"id":84043423,"identity":"9a9afe47-3a06-41e5-8f16-30d04758713b","added_by":"auto","created_at":"2025-06-06 06:41:47","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":595921,"visible":true,"origin":"","legend":"","description":"","filename":"STALARDSupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6782090/v1/d884ba2a4efbe56b94ddaf38.docx"},{"id":84043421,"identity":"09e6cec8-513a-4956-9022-d19630b9fb38","added_by":"auto","created_at":"2025-06-06 06:41:47","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12882,"visible":true,"origin":"","legend":"","description":"","filename":"STALARDSupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6782090/v1/9b1a3a8e7712b54f827741b0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"STALARD: Selective Target Amplification for Low-Abundance RNA Detection","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Low-abundance RNA, Reverse transcription-quantitative real-time PCR, Selective transcript enrichment","lastPublishedDoi":"10.21203/rs.3.rs-6782090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6782090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccurate quantification of transcript isoforms is critical for understanding gene regulation. However, conventional reverse transcription-quantitative real-time PCR (RT-qPCR) has limited sensitivity for low-abundance transcript isoforms, as quantification cycle (Cq) values above 30 are often considered unreliable. While transcriptome-wide analyses can address this limitation, they require costly deep sequencing and complex bioinformatics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo overcome the sensitivity limitations of conventional RT-qPCR for detecting low-abundance and alternatively spliced transcripts, we developed STALARD (Selective Target Amplification for Low-Abundance RNA Detection), a rapid (\u0026lt;2 hr) and targeted two-step RT-PCR method using standard laboratory reagents. STALARD selectively amplifies transcripts sharing a common 5′-end, enabling efficient quantification of low-abundance isoforms. 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Its compatibility with both qPCR and long-read sequencing makes it a versatile tool for analyzing transcript variants and identifying previously uncharacterized 3′-end structures.\u003c/p\u003e","manuscriptTitle":"STALARD: Selective Target Amplification for Low-Abundance RNA Detection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 06:41:42","doi":"10.21203/rs.3.rs-6782090/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T22:40:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-05T10:03:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282717653549993015621123008684993373712","date":"2025-07-02T08:34:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T22:58:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210515608432243250221716891605415195546","date":"2025-06-09T18:15:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T14:49:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-31T12:11:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-31T12:08:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Methods","date":"2025-05-30T07:13:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plme","sideBox":"Learn more about [Plant Methods](http://plantmethods.biomedcentral.com/)","snPcode":"13007","submissionUrl":"https://submission.nature.com/new-submission/13007/3","title":"Plant Methods","twitterHandle":"@PlantMethods","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9670f158-d9f6-4b84-9f4c-8e302b5e2f92","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:03:49+00:00","versionOfRecord":{"articleIdentity":"rs-6782090","link":"https://doi.org/10.1186/s13007-025-01443-z","journal":{"identity":"plant-methods","isVorOnly":false,"title":"Plant Methods"},"publishedOn":"2025-09-29 15:57:59","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-06-06 06:41:42","video":"","vorDoi":"10.1186/s13007-025-01443-z","vorDoiUrl":"https://doi.org/10.1186/s13007-025-01443-z","workflowStages":[]},"version":"v1","identity":"rs-6782090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6782090","identity":"rs-6782090","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}