A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown AlgaReportar como inadecuado




A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass 25-, 50- and 100 mg from strains of E. siliculosus RHO12; LIA4A; EC524 and REP10–11 isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer pH 9.5 which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants e.g. polysaccharides, polyphenols. The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA-RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass 0.195 and 0.284 µg mg−1 fresh weigh of RNA and DNA, respectively and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA-RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae.



Autor: Maria Greco , Claudio A. Sáez, Murray T. Brown, Maria Beatrice Bitonti

Fuente: http://plos.srce.hr/



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