A high-throughput method for quantifying transgene expression in transformed plants with real-time PCR analysis

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.