Product and process for isolating RNA
First Claim
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.
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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.
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Citations
7 Claims
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1. A method of isolating RNA from a biological tissue sample including RNA, DNA and proteins, comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6)
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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.
Specification