Poster: Education & outreach
Abs #
939: A DNAase-free, total RNA isolation method with ultra-low genomic DNA contamination applicable to a wide range of plant species and tissue types
Purification of total RNA from plant tissue has traditionally been hampered by the wide-range of plants and tissue types studied in the agricultural laboratory. Successful RNA isolations can suffer from several problematic components, including: nucleases; high polysaccharide content; waxes, oils, resins, and latexes; and other secondary metabolites. Purity, yield, and lack of genomic DNA are paramount to successful downstream applications. Popular isolation procedures include organic solvent extraction, or aqueous chaotropic extractions with silica-based solid phase devices for binding and purification. Although both of these methods have advantages and disadvantages DNAase treatment is required to reduce genomic DNA (gDNA) contamination prior to techniques such as real-time PCR or microarray analysis.
We have developed a rapid, qualitative and quantitative method for the isolation of plant RNA from a variety of species and tissue types that is virtually free of contaminating gDNA without the need for DNAase treatment. The method utilizes an aqueous extraction buffer in conjunction with two spin-columns; a prefiltration column that removes contaminating gDNA, cell debris and other contaminants followed by an RNA filter that captures the purified RNA. RNA yields and purity are equal to or higher than silica-based aqueous extraction methods or organic solvent extraction techniques. Absorbance ratios at 260/280 and 260/230 are above 1.8, indicating that the samples are free of protein and polysaccharide contamination, respectively. Electrophoretic analysis shows the RNA to be intact, with prominent rRNA bands. Quantitative PCR analysis of gDNA proves the isolated RNA has 1000 to 10,000 times less DNA contamination than typical organic solvent or silica based methods.
