Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Corydalis saxicola Under Various Abiotic Stresses

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Corydalis saxicola Under Various Abiotic Stresses | 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 Article Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Corydalis saxicola Under Various Abiotic Stresses Liang Kang, Zhaodi Wen, Han Liu, Ying Lu, Mei Qin, Lirong Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8096334/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 20 You are reading this latest preprint version Abstract Quantitative real-time PCR (qRT-PCR) is highly regarded for its exceptional sensitivity and specificity, making it a popular method for analyzing gene expression. The choice of suitable reference genes (RGs) is crucial for accurately assessing the dynamics of target gene expression through qRT-PCR. This study analyzes a set of eleven candidate RGs and focuses on identifying the most appropriate ones for examining gene expression in Corydalis saxicola across various tissues and under different abiotic stress conditions, including calcium (Ca 2+ ) stress, varying selenium (Se 2+ ) stresses, and distinct drought stresses. GAPDH2 and UBCE2-17 emerged as the most appropriate reference genes across various tissues of C. saxicola . In response to Ca 2+ stress, RPAP3 and UBCE2-5A demonstrated the highest expression stability. Under Se 2+ stress, GAPDH8 and α-TUB were identified as the optimal reference genes, while ACT4 and RPAP3 were deemed the best under drought conditions. Furthermore, GAPDH2 , TBP , and UBCE2-17 were confirmed as the most appropriate comprehensive reference genes applicable to different tissues and abiotic stress scenarios. To substantiate these findings, the expression profiles of CsBBEL9 and CsOMT9 , which are pivotal genes in the biosynthesis of benzylisoquinoline alkaloids, were analyzed across seven tissues and all stress treatments. The results indicated that employing stable reference genes resulted in consistent expression patterns, whereas the use of unstable reference genes caused artificially inflated expression levels of CsBBEL9 and CsOMT9 in stems, old leaves, samples treated with 25 mmol·L⁻¹ CaCl 2 , and those subjected to 12 days of drought stress, while leading to diminished expression levels under 5 mmol·L⁻¹ CaCl 2 and 50 µmol·L⁻¹ Na₂SeO₃ treatments. The varied expression profiles of target genes underscore the essential requirements for meticulous optimization of experimental conditions and careful selection of stable reference genes to guarantee reliable outcomes in gene expression research. This study has effectively identified the most suitable reference genes and their combinations for C. saxicola across various tissues and under multiple abiotic stress conditions, establishing a robust basis for further exploration of gene function and regulatory mechanisms in C. saxicola. Biological sciences/Biotechnology Biological sciences/Computational biology and bioinformatics Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Plant sciences real-time quantitative PCR reference genes Corydalis saxicola abiotic stresses gene expression Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.doc SupplementaryTables.xls Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 16 Feb, 2026 Reviews received at journal 12 Feb, 2026 Reviews received at journal 08 Feb, 2026 Reviews received at journal 08 Feb, 2026 Reviews received at journal 05 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviews received at journal 04 Feb, 2026 Reviewers agreed at journal 04 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers invited by journal 03 Feb, 2026 Editor assigned by journal 03 Feb, 2026 Editor invited by journal 16 Jan, 2026 Submission checks completed at journal 31 Dec, 2025 First submitted to journal 31 Dec, 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. 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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-8096334","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":585433482,"identity":"14dc863a-10c1-401f-8157-d7344e932939","order_by":0,"name":"Liang Kang","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants/Guangxi Key Laboratory of Conservation and Genetic Improvement of Medicinal Plant Resources","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Kang","suffix":""},{"id":585433483,"identity":"318c98c7-f8ad-4a7d-8390-48a351e9fbc4","order_by":1,"name":"Zhaodi 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reference genes, Corydalis saxicola, abiotic stresses, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-8096334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8096334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eQuantitative real-time PCR (qRT-PCR) is highly regarded for its exceptional sensitivity and specificity, making it a popular method for analyzing gene expression. The choice of suitable reference genes (RGs) is crucial for accurately assessing the dynamics of target gene expression through qRT-PCR. This study analyzes a set of eleven candidate RGs and focuses on identifying the most appropriate ones for examining gene expression in \u003cem\u003eCorydalis saxicola\u003c/em\u003e across various tissues and under different abiotic stress conditions, including calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) stress, varying selenium (Se\u003csup\u003e2+\u003c/sup\u003e) stresses, and distinct drought stresses. \u003cem\u003eGAPDH2\u003c/em\u003e and \u003cem\u003eUBCE2-17\u003c/em\u003e emerged as the most appropriate reference genes across various tissues of \u003cem\u003eC. saxicola\u003c/em\u003e. In response to Ca\u003csup\u003e2+\u003c/sup\u003e stress, \u003cem\u003eRPAP3\u003c/em\u003e and \u003cem\u003eUBCE2-5A\u003c/em\u003e demonstrated the highest expression stability. Under Se\u003csup\u003e2+\u003c/sup\u003e stress, \u003cem\u003eGAPDH8\u003c/em\u003e and \u003cem\u003eα-TUB\u003c/em\u003e were identified as the optimal reference genes, while \u003cem\u003eACT4\u003c/em\u003e and \u003cem\u003eRPAP3\u003c/em\u003e were deemed the best under drought conditions. Furthermore, \u003cem\u003eGAPDH2\u003c/em\u003e, \u003cem\u003eTBP\u003c/em\u003e, and \u003cem\u003eUBCE2-17\u003c/em\u003e were confirmed as the most appropriate comprehensive reference genes applicable to different tissues and abiotic stress scenarios. To substantiate these findings, the expression profiles of \u003cem\u003eCsBBEL9\u003c/em\u003e and \u003cem\u003eCsOMT9\u003c/em\u003e, which are pivotal genes in the biosynthesis of benzylisoquinoline alkaloids, were analyzed across seven tissues and all stress treatments. The results indicated that employing stable reference genes resulted in consistent expression patterns, whereas the use of unstable reference genes caused artificially inflated expression levels of \u003cem\u003eCsBBEL9\u003c/em\u003e and \u003cem\u003eCsOMT9\u003c/em\u003e in stems, old leaves, samples treated with 25 mmol\u0026middot;L⁻\u0026sup1; CaCl\u003csub\u003e2\u003c/sub\u003e, and those subjected to 12 days of drought stress, while leading to diminished expression levels under 5 mmol\u0026middot;L⁻\u0026sup1; CaCl\u003csub\u003e2\u003c/sub\u003e and 50 \u0026micro;mol\u0026middot;L⁻\u0026sup1; Na₂SeO₃ treatments. The varied expression profiles of target genes underscore the essential requirements for meticulous optimization of experimental conditions and careful selection of stable reference genes to guarantee reliable outcomes in gene expression research. This study has effectively identified the most suitable reference genes and their combinations for \u003cem\u003eC. saxicola\u003c/em\u003e across various tissues and under multiple abiotic stress conditions, establishing a robust basis for further exploration of gene function and regulatory mechanisms in \u003cem\u003eC. saxicola.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Corydalis saxicola Under Various Abiotic Stresses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 10:07:27","doi":"10.21203/rs.3.rs-8096334/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-16T07:21:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T07:31:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T04:01:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T02:34:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T19:53:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42716158808250111892667867555842634072","date":"2026-02-05T17:35:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-05T01:53:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151638364561929919445900043516860354250","date":"2026-02-04T05:29:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203724580989805328148925200680472586405","date":"2026-02-04T03:10:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131082187853724979449922803426775849077","date":"2026-02-04T02:53:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146435883030455055251122584266693140286","date":"2026-02-03T22:53:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51241022267274676680098396281002813930","date":"2026-02-03T19:05:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269379878993187340264659429261414244560","date":"2026-02-03T18:19:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53592333300658541554196564891988556191","date":"2026-02-03T17:18:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176406127523237508740850924543029417242","date":"2026-02-03T16:34:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-03T16:21:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-03T16:15:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-16T09:32:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-31T07:01:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-31T06:49:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"569e56a4-4d5a-46e8-882f-27a739422567","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62282216,"name":"Biological sciences/Biotechnology"},{"id":62282217,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":62282218,"name":"Biological sciences/Genetics"},{"id":62282219,"name":"Biological sciences/Molecular biology"},{"id":62282220,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-22T19:08:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 10:07:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8096334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8096334","identity":"rs-8096334","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}