All these data confirm the suitability of this protocol. Expression levels relative to ubiquitin UBQ; At5g are shown. As far as we know, all non-commercial protocols for RNA isolation from plant tissues use organic solvents during the first steps.
We present here a protocol that uses two simple buffers without organic solvents that includes a 30 min DNase digestion and requires only 90 min to obtain 24 DNA-free RNA samples. Although only data from leaves is presented here, RNA from most Arabidopsis tissues can be isolated since we have successfully used it with stems, roots and flowers data not shown and also from different vegetative tissues of the legume model plant Medicago truncatula Gao, personal communication.
The only tissues not amenable for the previous protocol were seeds and siliques and we streamlined and improved existing protocols that use organic extractions for RNA purification from those recalcitrant tissues. Altogether, our results indicate that the use of these protocols can be extended to other plant species. We determined that the RNA obtained at an intermediate step of the isolation protocol was of sufficient quality to allow DNase digestion before the final purification steps.
This modification shortens the protocol and maximizes yield bypassing the need to double precipitate the RNA.
After cDNA synthesis using oligo dT and qPCR using specific oligonucleotides annealing as far as possible from the polyA, we were able to detect and quantify the expression levels of both genes in all samples analyzed, confirming sensitivity and specificity. Accessibility to these protocols is ensured by its simplicity and low cost of the materials used.
With the increase of genomic approaches involving the manipulation of large numbers of samples, we feel these protocols will be extremely useful for plant scientists. Methods in Molecular Biology, Arabidopsis Protocols. Anal Biochem. United States Patent Plant Physiology.
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Yamaguchi S, Kamiya Y, Sun T: Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination. Plant J. Wobus U, Weber H: Seed maturation: genetic programmes and control signals.
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Curr Opin Plant Biol. Trends Plant Sci. Download references. We thank Inmaculada Gude and Elaine Smith for excellent technical assistance. We also thank Prof.
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Pilar Carbonero and Drs. Louise Thatcher and Hannetz Roschzttardtz for critical reading of the manuscript. LOS was responsible for experiments and supervision of this study and JVC contributed to the supervision. Be the first to like this. No Downloads. Views Total views.
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Description Details Customer Reviews The thale cress Arabidopsis thaliana is increasingly popular among plant scientists: it is small, easy to grow, and makes flowers, and the sequence of its small and simple genome was recently completed. This is the most complete and authoritative laboratory manual to be published on this model organism and the first to deal with genomic and proteomic approaches to its biology.
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