Plant Molecular Biology Reporter

, Volume 22, Issue 3, pp 289–300 | Cite as

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

  • Haibo Weng
  • Aihu Pan
  • Litao Yang
  • Chengmei Zhang
  • Zhili Liu
  • Dabing ZhangEmail author


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 (T0) 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.

Key words

Brassica napus HMG I/Y real-time PCR transgene copy number 



threshold cycle


cetyltrimethylammonium bromide

real-time PCR

real-time polymerase chain reaction


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Copyright information

© International Society for Plant Molecular Biology 2004

Authors and Affiliations

  • Haibo Weng
    • 1
    • 2
  • Aihu Pan
    • 3
  • Litao Yang
    • 1
  • Chengmei Zhang
    • 3
  • Zhili Liu
    • 1
  • Dabing Zhang
    • 2
    • 3
    Email author
  1. 1.Department of Biological Science and TechnologyNanjing UniversityNanjingPR China
  2. 2.School of Life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiPR China
  3. 3.Shanghai Key Laboratory of Agricultural Genetics and BreedingAgro-biotech Research Center, Shanghai Academy of Agricultural SciencesShanghaiPR China

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