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Quantitative transcript analysis in plants: improved first-strand cDNA synthesis.

Nai-Zhong Xiao·Lei Ba·Preben Bach Holm·Xing-Zhi Wang·Steve Bowra
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Abstract

The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived alpha-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of alpha-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

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Blotting, SouthernDNA, ComplementaryDNA, PlantPolymerase Chain ReactionReverse Transcriptase Polymerase Chain ReactionTranscription, GeneticTrifoliumTubulinJournal Article

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Nai-Zhong Xiao, Lei Ba, Preben Bach Holm, Xing-Zhi Wang, Steve Bowra. 2005. Quantitative transcript analysis in plants: improved first-strand cDNA synthesis.. https://doi.org/10.1111/j.1745-7270.2005.00052.x

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