Avtor/Urednik		Toplak, N; Okršlar, V; Stanič-Racman, D; Gruden, K; Žel, J
Naslov		A high-throughput method for quantifying transgene expression in transformed plants with real-time PCR analysis
Tip		članek
Vir		Plant Mol Biol
Vol. in št.		Letnik 22
Leto izdaje		2004
Obseg		str. 237-50
Jezik		eng
Abstrakt		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-YCR) are currently used for this purpose and enable qualitative and semiquantitativc estimation of transgene mRNA levels. We have introduced a real-time PCR method for quantitative determination of transgene expression Ievel 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 succesafully replece 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. ln addition, the described assay enables detection of the virus at increased sensitivity and reprodacibility and is therefore appropriate for use in routine PVYntn detection.
Deskriptorji		PLANTS, TRANSGENIC RNA, MESSENGER POTATOES TOBACCO GENE EXPRESSION POTYVIRUS BLOTTING, NORTHERN POLYMERASE CHAIN REACTION