Low-toxic and organic solvent-free isolation of RNA

Abstract

RNA is the central molecule that connects genetic makeup to the function of living cells and organisms. Since genes will also cease to be transcribed, RNA must decay so that the cellular RNA-pool can reflect the current transcriptional state. Undesired degradation is therefore a major challenge when RNA is isolated from living cells, tissues or organisms. Strongly protein-denaturing conditions, often involving toxic organic solvents, are usually employed to quickly inactivate RNases prior to separating nucleic acids from protein. We propose to combine high concentrations of urea, a compound used in fertilizers, with SDS as a non-volatile, low toxicity denaturant. The use of urea and SDS has previously been combined with organic solvent extraction, chaotropic salt-mediated column purification or selective precipitation of long RNA with LiCl. Here, we demonstrate that – akin to the classic alkaline lysis protocol for plasmid preparation – addition of potassium acetate forms insoluble potassium dodecyl sulfate, which can be removed along with denatured proteins and part of the genomic DNA by centrifugation. The RNA is then precipitated with isopropanol, recovering also small non-coding RNAs. We demonstrate that these somewhat crude RNA preparations are nonetheless sufficiently clean to be quantified via classic UV absorption measurements, stable over the time-course of a typical molecular biology reaction and do not harbor inhibitors of reverse transcriptase. While it seems unlikely that we are the first to conceive this simple strategy, we are not aware of any corresponding prior publication.