Method for isolating RNA including small RNA with high yield

A method for isolating RNA, including small RNA having a length of 200 nt or less, from a sample, comprising the following steps:

• a) providing a composition comprising RNA and a chaotropic agent;
• b) adding alcohol to generate a first mixture;
• c) incubating the first mixture for at least 2 min to generate an incubated first mixture;
• d) adding additional alcohol to the incubated first mixture to generate a second mixture so that the overall alcohol concentration in the second mixture is ≥50%;
• e) binding RNA including small RNA to a nucleic acid binding solid phase, wherein the nucleic acid binding solid phase is contacted with the second mixture in step d), and optionally additionally with one or more selected from the group consisting of (i) the composition in step a), (ii) the first mixture in step b), and (iii) the first incubated mixture in step c);
• f) optionally washing the bound RNA;
• and
• g) optionally eluting RNA from the solid phase;
• wherein no separation of the nucleic acid binding solid phase, if present at steps b), c) and/or d), and the first mixture, the incubated first mixture, or the second mixture is performed at any of steps b), c) and d),
• wherein the alcohol that is added in step b) and in step d) is selected from methanol, ethanol, propanol, isopropanol, butanol, and mixtures thereof, and
• wherein
• i) the amount of alcohol added in step b) corresponds to approximately 40% to 80% of the overall alcohol concentration that is added for binding in step e);
• and/or
• ii) an amount of alcohol is added in step b) so that the first mixture comprises the alcohol in a concentration that lies in a range from 25% (v/v) to 45% (v/v).