Reagents and methods for isolation of purified RNA
OwnerPiotr Chomczynskiinventor
Life of this patent
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Abstract
Compositions and methods to isolate intact RNA that is substantially free of DNA, termed purified RNA. RNA from any source (e.g., human, other animals, plants, viruses, etc.) may be isolated. In one embodiment, the sample is treated with phenol at a pH less than 4.0 and purified RNA is recovered from the aqueous phase. In another embodiment, RNA is precipitated from an acidified sample containing a low volume of an organic solvent. Other embodiments are disclosed. The same inventive composition may be used for several embodiments with pH adjustment. Purified RNA obtained by the inventive method may be used in assays where DNA contamination is undesirable, such as the polymerase chain reaction.
Claims61
A phenol-containing composition for isolating purified RNA comprising phenol at a final concentration ranging from about 3%w/w to about 98%w/w and a buffer sufficient to maintain a pH of the final composition in the range from about pH 3.6 to below pH 4.0.
A phenol-containing composition for isolating purified RNA comprising phenol at a final concentration ranging from about 3%w/w to less than 30%w/w, and a buffer sufficient to maintain a pH of the final composition in the range from about pH 3.9 to about pH 5.5.
A phenol-free aqueous composition for isolating purified RNA, the phenol-free composition comprising at least one ribonuclease inhibitor and a buffer selected from at least one of acetate, citrate, phosphate, phthalate, tartrate, lactate, or mixtures thereof, sufficient to maintain a pH of the composition in the range from about pH 3.6 to below pH 4.0.
A phenol-free phase separation composition for use in isolating purified RNA by phase separation comprising at least one hydrophobic organic solvent at a final concentration in the range from about 10%w/w to about 40%w/w, and at least one acid sufficient to maintain a pH in the range of about pH 3.6 to below pH 4.0 during phase separation, and an optional acid solubilizer.
A method for isolating purified RNA from a biological sample comprising
- a) treating the sample with a reagent comprising phenol at a final concentration ranging from about 10%w/w to about 60%w/w and at least one ribonuclease inhibitor,
- b) mixing the sample with at least one hydrophobic solvent while maintaining a pH in the range from about pH 3.6 to below pH 4.0,
- c) recovering purified RNA from an aqueous phase to which about an equal volume of a water-soluble organic solvent is added to precipitate the purified RNA, and
- d) washing and solubilizing the precipitated RNA.
A method for isolating purified RNA from a biological sample comprising the steps of
- a) treating the sample with a reagent comprising phenol at a final concentration ranging from about 3%w/w to less than 30%w/w and a buffer sufficient to maintain a pH of the composition in the range from about pH 3.6 to about pH 5.5,
- b) sedimenting or filtering the sample to obtain a purified sample substantially free of DNA, proteins, and cellular components,
- c) adding to the purified sample about an equal volume of a water-soluble organic solvent to precipitate purified RNA,
- d) sedimenting or filtering the precipitated RNA, and
- e) washing and solubilizing the precipitated RNA.
A two-step method for isolating purified RNA from a biological sample comprising
- a) treating the sample with a reagent comprising phenol at a final concentration ranging from about 3%w/w to less than 30%w/w, at least one chaotrope, and a buffer sufficient to maintain a pH of the composition in the range from about pH 3.6 to about pH 5.5,
- b) sedimenting or filtering the sample to obtain a purified sample substantially free of DNA, proteins, and cellular components,
- c) adding to the purified sample at least one hydrophobic organic solvent and a buffer in a concentration sufficient to maintain a pH of the purified sample in the range from about pH 3.6 to below pH 4.0,
- d) recovering purified RNA from an aqueous phase to which about an equal volume of a water soluble organic solvent is added to precipitate purified RNA,
- e) sedimenting or filtrating the precipitated RNA, and
- f) washing and solubilizing the precipitated RNA.
A method for isolating purified RNA from an RNA- and salt-containing solution comprising adjusting a pH of the solution with a buffer in an amount sufficient to result in a maximum pH of 3.3, thereafter precipitating the purified RNA.
A method for selectively precipitating higher molecular weight RNA from a biological sample comprising
- treating the sample with an aqueous composition comprising phenol at a final concentration ranging from about 1%w/w to about 60%w/w, at least one chaotrope, a buffer in a concentration sufficient to maintain a pH of the composition in the range from about pH 2.0 to about pH 9.0, at least one water-soluble organic solvent at a concentration from about 10%w/w to about 40%w/w to selectively precipitate higher molecular weight RNA from the sample, and
- precipitating purified higher molecular weight RNA from the sample.
Dependencies are inferred from the claim text.