Dimethyl sulfoxide improves sensitivity and specificity of RT-PCR and qRT-PCR amplification of low-expressed transgenes
The expression of transgenes in a host plant may be low for a number of reasons. Both low expression and poor specificity of amplification were encountered during analysis of the expression of the Arabidopsis cytokinin oxidase/dehydrogenase (AtCKX1) gene in transgenic Centaurium erythraea. The o...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
University of Belgrade, University of Novi Sad
2012-01-01
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| Series: | Archives of Biological Sciences |
| Subjects: | |
| Online Access: | http://www.doiserbia.nb.rs/img/doi/0354-4664/2012/0354-46641203865S.pdf |
| Summary: | The expression of transgenes in a host plant may be low for a number of reasons. Both low expression and poor specificity of amplification were encountered during analysis of the expression of the Arabidopsis cytokinin oxidase/dehydrogenase (AtCKX1) gene in transgenic Centaurium erythraea. The optimization of the PCR protocol involved a gradient of annealing temperatures, as well as the application of seven PCR enhancers: formamide, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, trehalose, BSA and Tween-20. The best results for AtCKX1 amplification were obtained at 55.1ºC, with the addition of 5% DMSO. Glycerol and trehalose also improved the sensitivity of amplification, while formamide, ethylene glycol and BSA enhanced only the amplification of control purified targets, but not the transcripts. Tween-20 inhibited PCR. DMSO enhanced AtCKX1 PCR amplification and improved the specificity of qPCR amplification, as well as the assay reproducibility. This work emphasizes the usefulness of additives, which are rarely used for PCR optimization in real-time experiments.[Acknowledgments. The research presented here was funded by the Ministry of Education and Science of the Republic of Serbia (grants Nos. ON173015 and ON173024).] |
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| ISSN: | 0354-4664 1821-4339 |