Skip to main content

Advertisement

SpringerLink
Log in

Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction

  • Technical report
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCyclerTM system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCyclerTM software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • B Bubner IT Baldwin (2004) ArticleTitleUse of real-time PCR for determining copy number and zygosity in transgenic plants Plant Cell Rep 23 263–271 Occurrence Handle10.1007/s00299-004-0859-y Occurrence Handle1:CAS:528:DC%2BD2cXovVCms7g%3D Occurrence Handle15368076

    Article  CAS  PubMed  Google Scholar 

  • B Bubner K Gase IT Baldwin (2004) ArticleTitleTwo-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR BMC Biotech 4 14

    Google Scholar 

  • CJ Fregeau RA Aubin JC Elliott SS Gill RM Fourney (1995) ArticleTitleCharacterization of human lymphoid cell lines GM9947 and GM9948 as intra- and interlaboratory reference standards for DNA typing Genomics 28 184–197 Occurrence Handle1:CAS:528:DyaK2MXnsVOntL0%3D Occurrence Handle8530025

    CAS  PubMed  Google Scholar 

  • MA German M Kandel-Kfir D Swarzberg T Matsevitz D Granot (2003) ArticleTitleA rapid method for the analysis of zygosity in transgenic plants Plant Sci 164 183–187 Occurrence Handle10.1016/S0168-9452(02)00381-3 Occurrence Handle1:CAS:528:DC%2BD3sXivVCmsQ%3D%3D

    Article  CAS  Google Scholar 

  • H Kaminaka S Morita M Nakajima T Masumura T Kunisuke (1998) ArticleTitleGene cloning and expression of cytosolic glutathione reductase in rice (Oryza Sativa L.) Plant Cell Physiol 39 IssueID12 1269–1280 Occurrence Handle1:CAS:528:DyaK1MXhsFyitQ%3D%3D Occurrence Handle10050312

    CAS  PubMed  Google Scholar 

  • KJ Livak TD Schmittgen (2001) ArticleTitleAnalysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method Methods 25 402–408 Occurrence Handle10.1006/meth.2001.1262 Occurrence Handle1:CAS:528:DC%2BD38XhtFelt7s%3D Occurrence Handle11846609

    Article  CAS  PubMed  Google Scholar 

  • G Mason P Provero AM Vaira GP Accotto (2002) ArticleTitleEstimating the number of integrations in transformed plants by quantitative real-time PCR BMC Biotech 2 20

    Google Scholar 

  • MA Schmidt WA Parrott (2001) ArticleTitleQuantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction Plant Cell Rep 20 422–428 Occurrence Handle1:CAS:528:DC%2BD3MXltFOhurY%3D

    CAS  Google Scholar 

  • RR Spaete RM Thayer WS Probert FR Masiarz SH Chamberlain L Rasmussen TC Merigan C Pachl (1988) ArticleTitleHuman cytomegalovirus strain Towne glycoprotein B is processed by proteolytic cleavage Virology 167 IssueID1 207–225 Occurrence Handle10.1016/0042-6822(88)90071-2 Occurrence Handle1:CAS:528:DyaL1MXkslajsro%3D Occurrence Handle2460994

    Article  CAS  PubMed  Google Scholar 

  • Tackaberry E, Dudani A, Sardana R, Altosaar I and Ganz P (2002) Production of therapeutic proteins in transgenic cereal crops Patent Application (2,400,887 (Canada) and (10/230,365 (United States) August 29, 2002

  • L Tesson J-M Heslan S Menoret I Anegon (2002) ArticleTitleRapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR Transgenic Res 11 43–48 Occurrence Handle10.1023/A:1013928600442 Occurrence Handle1:CAS:528:DC%2BD38Xit1Cmtbc%3D Occurrence Handle11874102

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Sir Frederick Banting Research Centre, Tunney’s Pasture, A/L 2201C, K1A 0L2, Ottawa, Ontario, Canada

    Fiona A. Prior, Eilleen S. Tackaberry, Rémy A. Aubin & William L. Casley

  2. Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada

    Eilleen S. Tackaberry & Rémy A. Aubin

  3. Department of Biology, University of Ottawa, Ottawa, Ontario, Canada

    William L. Casley

Authors
  1. Fiona A. Prior
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eilleen S. Tackaberry
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rémy A. Aubin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. William L. Casley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William L. Casley.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Prior, F.A., Tackaberry, E.S., Aubin, R.A. et al. Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction. Transgenic Res 15, 261–265 (2006). https://doi.org/10.1007/s11248-005-4024-3

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-005-4024-3

Keywords

Search

Navigation