NDLI: Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay withHMG I/Y as an endogenous reference gene

Content Provider	Springer Nature Link
Author	Weng, Haibo Pan, Aihu Yang, Litao Zhang, Chengmei Liu, Zhili Zhang, Dabing
Copyright Year	2004
Abstract	In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T$_{0}$) were calculated by comparing threshold cycle (C $_{T}$) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC $_{T}$ value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.
Starting Page	289
Ending Page	300
Page Count	12
File Format	PDF
ISSN	07359640
Journal	Plant Molecular Biology Reporter
Volume Number	22
Issue Number	3
e-ISSN	15729818
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-09-03
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Brassica napus HMG I/Y real-time PCR transgene copy number Plant Sciences Plant Breeding/Biotechnology Proteomics Metabolomics Bioinformatics
Content Type	Text
Resource Type	Article
Subject	Plant Science Molecular Biology

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