Abstract
When analysing plant transformation in plant transgenic lines, determining the level of transgene expression is essential. Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR) are currently used for this purpose and enable qualitative and semiquantitative estimation of transgene mRNA levels. We have introduced a real-time PCR method for quantitative determination of transgene expression level in transgenic potato plants containing the gene for coat protein (CP) of potato virus Y strain NTN (PVYNTN) in order to provide a reliable, high-throughput method that could successfully replace the Northern blot analysis. The method has been compared with other available methods for gene expression analysis with respect to accuracy, sensitivity, specificity, and throughput. The effectiveness of the real-time PCR assay was confirmed on transgenic tobacco plants. It proved to be accurate, sensitive rapid, and sufficiently reproducible for further application in high-throughput molecular characterisation of transgenic lines. In addition, the described assay enables detection of the virus at increased sensitivity and reproducibility and is therefore appropriate for use in routine PVYNTN detection.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CP:
-
coat protein
- CT :
-
cycle threshold
- luc :
-
luciferase
- PVYNTN :
-
potato virus Y strain NTN
- RT-PCR:
-
reverse-transcription polymerase chain reaction
References
Alwine JC, Kemp DJ, and Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Nat Acad Sci 74: 5350–5354.
Applied Biosystems (1997) User Bulletin #2, ABI PRISM 7700 Sequence Detection System. User Bulletin. Applied Biosystems, Foster City, USA.
Brodmann PD, Ilg EC, Berthoud H, and Herrmann A (2002) Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food. J AOAC Int 85(3): 646–653.
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29: 23–39.
Czechowski T, Bari RP, Stitt M, Scheible WR, and Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38(2): 366–379.
Dean JD, Goodwin PH, and Hsiang T (2002) Comparison of relative RT-PCR and Northern blot analyses to measure expression of β-1,3-glucanase inNicotiana benthamiana infected withColltotrichum destructivum. Plant Mol Biol Rep 20: 347–356.
Fabre F, Kervarrec C, Mieuzet L, Riault G, Vialatte A, and Jacquot E (2003) Improvement ofBarley yellow dwarf virus-PAV detection in single aphids using a fluorescent real time RT-PCR. J Virol Methods 110(1): 51–60.
Freeman WM, Walker SJ, and Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. BioTechniques 26: 112–125.
Gattei V, Degan M, De Iuliis A, Rossi FM, Aldinucci D, and Pinto A (1997) Competetive reverse-transcriptase PCR: a useful alternative to Northern blotting for quantitative estimation of relative abundances of specific mRNA in precious samples. Biochem J Lett 325: 565–567.
German MA, Kandel-Kfir M, Swarzberg D, Matsevitz T, and Granot D (2002) A rapid method for the analysis of zygosity in transgenic plants. Plant Sci 164: 183–187.
Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30: 503–512.
Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, and Mathieu C (2001) An overview of real-time quantitative PCR application to quantify cytokine gene expression. Methods 25: 386–401.
Heid CA, Stevens J, Livak KJ, and Williams PM (1996) Real-time quantitative PCR. Genome Res 6: 986–994.
Holland PM, Abramson RD, Watson R, and Gelfand DH (1991) Detection of a specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity ofThermus aquaticus DNA polymerase. Proc Nat Acad of Sci 88: 7276–7280.
Ingham DJ, Beer S, Money S, and Hansen G (2001) Quantitative real-time PCR assay for determining transgene copy number in transformed plants. BioTechniques 31: 132–140.
Kim BR, Nam HY, Kim SU, Kim SI, and Chang YJ (2003) Normalization of reverse transcription quantitative-PCR with house keeping genes in rice. Biotechnol Lett 25 (21): 1869–1872.
Klein D (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8: 257–260.
Leutenegger CM, Mislin CN, Sigrist B, Ehrengruber MU, Hofmann-Lehmann R, and Lutz H (1999) Quantitative real-time PCR for the measurement of feline cytokine mRNA. Vet Immunol Immunopathol 71: 291–305.
Logemann J (1987) Improved method for the isolation of RNA from plant-tissues. Anal Biochem 163(1): 16–20.
Lovatt A (2002) Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products. J Biotechnol 82: 279–300.
Mackay IM, Arden KE, and Nitsche A (2002) Real-time PCR in virology. Nucleic Acids Res 30: 1292–1305.
Miyamoto T, Nakamura T, Nagao I, and Obokata J (2000) Quantitative analysis of transiently expressed mRNA in particle-bombarded tobacco seedlings. Plant Mol Biol Rep 18: 101–107.
Myslik JT and Nassuth A (2001) Rapid detection of viruses, transgenes, and mRNAs in small plant leaf samples. Plant Mol Biol Rep 19: 329–340.
Schaart JG, Salentijn EMJ, and Krens FA (2002) Tissue-specific expresion of the β-glucuronidase gene in transgenic strawberry (Fragaria ×ananassa) plants. Plant Cell Reports 21: 313–319.
Schmidt MA and Parrott WA (2001) Quantitative detection of transgenes in soybean (Glycine max (L.) Merrill) and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction. Plant Cell Reports 20: 422–428.
Singh RP (1998) Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids. J Virol Methods 74: 125–138.
Singh M and Singh RP (1998) Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization. J Virol Methods 60: 47–57.
Song P, Cai CQ, Skokut M, Kosegi BD, and Petolino JF (2002) Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS-derived transgenic maize. Plant Cell Reports 20: 948–954.
Stanič-Racman D, Mcgeachy K, Reavy B, Štrukelj B, Žel J, and Barker H (2001) Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from NTN isolate of Potato virus Y. Ann Appl Biol 139(2): 269–275.
Stanič-Racman D, Štrukelj B, Štrucl R, Demšar T, Reavy B, Barker H, and Žel J (1999) Transformation ofNicotiana tabacum cv. Samsun by the Coat Protein Gene of PVYNTN. Phyton 39: 271–276.
Yano A, Kaneko N, Ida H, Yamaguchi T, and Hanada N (2002) Real-time PCR for quantification ofStreptococcus mutans. FEMS Microbiol Lett 217: 23–30.
Yongbaek K, Gharaibeh SM, Stedman NL, and Brown TP (2002) Comparison and vertification of quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) and real time RT-PCR for avian leucosis virus subgroup J. J Virol Methods 102: 1–8.
Wall SJ and Edwards DR (2002) Quantitative reverse transcription-polymerase chain reaction (RT-PCR): a comparison of primer-dropping, competitive, and real-time RT-PCRs. Anal Biochem 300: 269–273.
Whitcombe D, Theaker J, Guy SP, Brown T, and Little S (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 17: 804–807.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Toplak, N., Okršlar, V., Stanič-Racman, D. et al. A high-throughput method for quantifying transgene expression in transformed plants with real-time PCR analysis. Plant Mol Biol Rep 22, 237–250 (2004). https://doi.org/10.1007/BF02773134
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF02773134