Product and process for isolating RNA

• US 4,843,155 A
• Filed: 11/19/1987
• Issued: 06/27/1989
• Est. Priority Date: 11/19/1987
• Status: Expired due to Term

• Current Assignee: Chomczynski, Piotr
• Original Assignee: Chomczynski, Piotr
• Inventors: Chomczynski, Piotr
• Primary Examiner(s): Griffin, Ronald W.
• Assistant Examiner(s): Crane, L. Eric
• Application Number:
  US07/123,107
• Time in Patent Office: 586 Days
• Field of Search: 536/27, 536/28, 536/29, 536/24
• US Class Current: 536/25.41
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Abstract

The present invention discloses a novel method for isolating RNA from biological tissue samples and a novel solvent adapted for use in the disclosed method. The method employs a single extraction using the solvent containing guanidinium and phenol. The solvent is stable for about one month at room temperature without any appreciable phenol oxidation or decomposition. Application of the disclosed method and solvent to a biological tissue sample results in the isolation of a high yield of RNA in a substantially pure and undegraded form. The whole procedure can be completed in three hours, much more quickly than other procedures.

Claims

1. A method of isolating RNA from a biological tissue sample including RNA, DNA and proteins, comprising:

homogenizing said tissue sample with a denaturing solvent which consists essentially of a guanidinium compound selected from the group consisting of guanidinium thiocyanate and guanidinium chloride, phenol and a buffer in an amount sufficient to maintain said solvent at or about pH 4, to form a homogenate;

adding a water-insoluble organic solvent to said homogenate and centrifuging to form a two-phase mixture having an aqueous phase and an organic phase, wherein said RNA is concentrated in said aqueous phase, and said DNA and proteins are concentrated in said organic phase or in the interphase;

precipitating said RNA from said aqueous phase by adding a lower alcohol to said aqueous phase; and

recovering the precipitated RNA by centrifugation of said aqueous phase and decanting the supernatant liquid.

2. A method of claim 1 wherein said denaturing solvent is stored for more than about four weeks at substantially room temperature before use in said method.

3. The method of claim 1 wherein said tissue sample is homogenized in said solvent in the proportion of about 2ml solvent/100 mg. tissue sample.

4. The method of claim 1 wherein said water-insoluble organic solvent is added to said homogenate in an amount equal to approximately 1/10 the volume of the homogenate.

5. The method of claim 1 wherein said lower alcohol is added in approximately equal volume to said aqueous phase.

6. The method of claim 1 including the further step of washing the precipitated RNA in ethanol and drying it.

7. A solvent, which remains stable after prolonged storage, for extracting RNA from biological tissue when said solvent is added to the tissue, said solvent consisting essentially of:

a solution of a guanidinium compound selected from the group consisting of guanidinium thiocyanate and guanidinium chloride, phenol and a buffer in an amount sufficient to maintain said solvent at or about PH 4, said solution present in a proportion which is effective both to substantially reduce oxidation and decomposition of said phenol and thereby to maintain said solvent stable during storage in comparison to phenol stored alone, and, in conjunction with said phenol, to separate RNA from said biological tissue, said proportion of said solution making up from about 40% to about 60% of said solvent by volume.