{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T15:48:30Z","timestamp":1784130510937,"version":"3.55.0"},"reference-count":18,"publisher":"Springer Science and Business Media LLC","issue":"S7","license":[{"start":{"date-parts":[[2007,11,1]],"date-time":"2007-11-01T00:00:00Z","timestamp":1193875200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/2.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Bioinformatics"],"published-print":{"date-parts":[[2007,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1471-2105-8-s7-s6","type":"journal-article","created":{"date-parts":[[2007,11,1]],"date-time":"2007-11-01T19:14:16Z","timestamp":1193944456000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":50,"title":["Statistical tools for transgene copy number estimation based on real-time PCR"],"prefix":"10.1186","volume":"8","author":[{"given":"Joshua S","family":"Yuan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Burris","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan R","family":"Stewart","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayalew","family":"Mentewab","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr","given":"C Neal","family":"Stewart","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2007,11,1]]},"reference":[{"issue":"4691","key":"1937_CR1","doi-asserted-by":"publisher","first-page":"1229","DOI":"10.1126\/science.227.4691.1229","volume":"227","author":"RBF Horsch","year":"1985","unstructured":"Horsch RBF, Hoffmann JE, Eichholtz NL, Rogers D, Fraley SG, R T: A simple and general method for transferring genes into plants. Science 1985,227(4691):1229\u20131231. 10.1126\/science.227.4691.1229","journal-title":"Science"},{"issue":"2","key":"1937_CR2","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1016\/j.jbbm.2005.05.006","volume":"64","author":"D Mitrecic","year":"2005","unstructured":"Mitrecic D, Huzak M, Curlin M, Gajovic S: An improved method for determination of gene copy numbers in transgenic mice by serial dilution curves obtained by real-time quantitative PCR assay. J Biochem Biophys Methods 2005,64(2):83\u201398. 10.1016\/j.jbbm.2005.05.006","journal-title":"J Biochem Biophys Methods"},{"key":"1937_CR3","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/S0076-6879(04)92001-0","volume":"392","author":"K McGinnis","year":"2005","unstructured":"McGinnis K, Chandler V, Cone K, Kaeppler H, Kaeppler S, Kerschen A, Pikaard C, Richards E, Sidorenko L, Smith T, et al.: Transgene-induced RNA interference as a tool for plant functional genomics. Methods Enzymol 2005, 392: 1\u201324.","journal-title":"Methods Enzymol"},{"issue":"3","key":"1937_CR4","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1093\/jxb\/erl228","volume":"58","author":"W Tang","year":"2007","unstructured":"Tang W, Newton RJ, Weidner DA: Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 2007,58(3):545\u2013554. 10.1093\/jxb\/erl228","journal-title":"J Exp Bot"},{"key":"1937_CR5","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1186\/1472-6750-4-14","volume":"4","author":"B Bubner","year":"2004","unstructured":"Bubner B, Gase K, Baldwin IT: Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR. BMC Biotechnol 2004, 4: 14. 10.1186\/1472-6750-4-14","journal-title":"BMC Biotechnol"},{"issue":"5","key":"1937_CR6","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1007\/s00299-004-0859-y","volume":"23","author":"B Bubner","year":"2004","unstructured":"Bubner B, Baldwin IT: Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep 2004,23(5):263\u2013271. 10.1007\/s00299-004-0859-y","journal-title":"Plant Cell Rep"},{"issue":"9","key":"1937_CR7","doi-asserted-by":"publisher","first-page":"1177","DOI":"10.1038\/nbt1134","volume":"23","author":"A Mentewab","year":"2005","unstructured":"Mentewab A, Stewart CN Jr: Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants. Nat Biotechnol 2005,23(9):1177\u20131180. 10.1038\/nbt1134","journal-title":"Nat Biotechnol"},{"issue":"1","key":"1937_CR8","doi-asserted-by":"crossref","first-page":"132","DOI":"10.2144\/01311rr04","volume":"31","author":"DJ Ingham","year":"2001","unstructured":"Ingham DJ, Beer S, Money S, Hansen G: Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 2001,31(1):132\u2013134. 136\u2013140. 136\u2013140.","journal-title":"Biotechniques"},{"issue":"3","key":"1937_CR9","first-page":"279","volume":"82","author":"A Lovatt","year":"2002","unstructured":"Lovatt A: Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products. J Biotechnol 2002,82(3):279\u2013300.","journal-title":"J Biotechnol"},{"key":"1937_CR10","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1186\/1472-6750-2-20","volume":"2","author":"G Mason","year":"2002","unstructured":"Mason G, Provero P, Vaira AM, Accotto GP: Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2002, 2: 20. 10.1186\/1472-6750-2-20","journal-title":"BMC Biotechnol"},{"issue":"4","key":"1937_CR11","doi-asserted-by":"crossref","first-page":"610","DOI":"10.2144\/04374ST06","volume":"37","author":"M Ballester","year":"2004","unstructured":"Ballester M, Castello A, Ibanez E, Sanchez A, Folch JM: Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals. Biotechniques 2004,37(4):610\u2013613.","journal-title":"Biotechniques"},{"issue":"10\u201311","key":"1937_CR12","doi-asserted-by":"publisher","first-page":"759","DOI":"10.1007\/s00299-004-0881-0","volume":"23","author":"L Yang","year":"2005","unstructured":"Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D: Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep 2005,23(10\u201311):759\u2013763. 10.1007\/s00299-004-0881-0","journal-title":"Plant Cell Rep"},{"issue":"2","key":"1937_CR13","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1007\/s11248-005-4024-3","volume":"15","author":"FA Prior","year":"2006","unstructured":"Prior FA, Tackaberry ES, Aubin RA, Casley WL: Accurate determination of zygosity in transgenic rice by real-time PCR does not require standard curves or efficiency correction. Transgenic Res 2006,15(2):261\u2013265. 10.1007\/s11248-005-4024-3","journal-title":"Transgenic Res"},{"key":"1937_CR14","volume-title":"Biotechnology Journal","author":"JS Yuan","year":"2008","unstructured":"Yuan JS, Wang D, Stewart CN Jr: Statistical methods for efficiency adjusted real-time PCR analysis. Biotechnology Journal 2008, in press."},{"key":"1937_CR15","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1186\/1471-2105-7-85","volume":"7","author":"JS Yuan","year":"2006","unstructured":"Yuan JS, Reed A, Chen F, Stewart CN Jr: Statistical analysis of real-time PCR data. BMC Bioinformatics 2006, 7: 85. 10.1186\/1471-2105-7-85","journal-title":"BMC Bioinformatics"},{"issue":"2","key":"1937_CR16","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1007\/BF02772725","volume":"22","author":"ZW Li","year":"2004","unstructured":"Li ZW, Hansen JL, Liu Y, Zemetra RS, Berger PH: Using real-time PCR to determine transgene copy number in wheat. Plant Molecular Biology Reporter 2004,22(2):179\u2013188.","journal-title":"Plant Molecular Biology Reporter"},{"issue":"6","key":"1937_CR17","doi-asserted-by":"crossref","first-page":"990","DOI":"10.2144\/04376BIN02","volume":"37","author":"P Cook","year":"2004","unstructured":"Cook P, Fu C, Hickey M, Han ES, Miller KS: SAS programs for real-time RT-PCR having multiple independent samples. Biotechniques 2004,37(6):990\u2013995.","journal-title":"Biotechniques"},{"issue":"3","key":"1937_CR18","doi-asserted-by":"publisher","first-page":"473","DOI":"10.1111\/j.1399-3054.1962.tb08052.x","volume":"15","author":"T Murashige","year":"1962","unstructured":"Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962,15(3):473\u2013497. 10.1111\/j.1399-3054.1962.tb08052.x","journal-title":"Physiol Plant"}],"container-title":["BMC Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/link.springer.com\/content\/pdf\/10.1186\/1471-2105-8-S7-S6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/link.springer.com\/article\/10.1186\/1471-2105-8-S7-S6\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/1471-2105-8-S7-S6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T21:19:13Z","timestamp":1630444753000},"score":1,"resource":{"primary":{"URL":"https:\/\/bmcbioinformatics.biomedcentral.com\/articles\/10.1186\/1471-2105-8-S7-S6"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2007,11,1]]},"references-count":18,"journal-issue":{"issue":"S7","published-print":{"date-parts":[[2007,12]]}},"alternative-id":["1937"],"URL":"https:\/\/doi.org\/10.1186\/1471-2105-8-s7-s6","relation":{},"ISSN":["1471-2105"],"issn-type":[{"value":"1471-2105","type":"electronic"}],"subject":[],"published":{"date-parts":[[2007,11,1]]},"assertion":[{"value":"1 November 2007","order":1,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"S6"}}