Abstract
Shelf-stable solvent solutions and methods for simultaneously isolating RNA, DNA and proteins from biological samples are disclosed. The solvent solutions include phenol and a guanidinium compound, preferably at a concentration below about 2M, which is effective in isolating substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from the same biological sample.
Patent number: 5346994Filing date: Jan 28, 1992
Issue date: Sep 13, 1994
Inventor: Piotr Chomczynski
download
What is claimed is:
1. A solvent solution comprising: effective amounts of phenol, a guanidinium compound and a thiocyanate compound selected from the group consisting of ammonium thiocyanate and sodium thiocyanate for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue.
2. The solvent solution of claim 1, said thiocyanate compound being present at a concentration in the range of about 0.1-0.6M, based on the total volume of said solvent solution.
3. A solvent solution comprising: effective amounts of phenol, a guanidinium compound and sodium acetate present at a concentration of about 0.1M, based on the total volume of said solvent solution, said solvent solution having a pH of about 5.0 for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue.
4. A solvent solution comprising: effective amounts of phenol, a guanidium compound and a phenol solubilizer for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue.
5. The solvent solution of claim 4, said phenol solubilizer being glycerol.
6. The solvent solution of claim 5, said glycerol being present in the range of about 3%-10% by volume of said solvent solution, based on the total volume of said solvent solution.
7. The solvent solution of claim 6, said phenol being present in the range of about 30%-50% by volume of said solvent solution, based on the total volume of said solvent solution.
8. A solvent solution for extracting substantially pure RNA, DNA and proteins from biological tissue, said solvent solution comprising:
- (a) guanidinium thiocyanate at a concentration in the range of about 0.5-2M, based on the total volume of said solvent solution;
- (b) a buffer in an amount sufficient to maintain the pH of said solvent solution in the range of about 4-6;
- (c) phenol in the amount of about 30%-50% by volume based on the total volume of said solvent solution; and
- (d) a phenol solubilizer in the amount of about 3%-10% by volume based on the total volume of said solvent solution.
9. The solvent solution of claim 8 further comprising ammonium thiocyanate at a concentration in the range of about 0.1-0.6M, based on the total volume of said solvent solution.
10. The solvent solution of claim 8, said phenol solubilizer being glycerol.
11. The solvent solution of claim 8, said guanidinium thiocyanate concentration being about 0.8M.
12. The solvent solution of claim 9, said ammonium thiocyanate concentration being about 0.4M.
13. The solvent solution of claim 8, said buffer being sodium acetate.
14. The solvent solution of claim 13, said sodium acetate being present at a concentration of about 0.1M, based on the total volume of said solvent solution, said solvent solution having a pH of about 5.0.
15. The solvent solution of claim 10, said glycerol comprising about 5% by volume of said solvent solution.
16. The solvent solution of claim 8, said phenol comprising about 38% by volume of said solvent solution.
17. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:
- (a) homogenizing a tissue sample in the solvent solution of claim 1 to form a homogenate;
- (b) adding a water-insoluble organic solvent to said homogenate and sedimenting to form a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA;
- (c) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;
- (d) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and
- (e) recovering DNA from the interphase by washing the interphase with a predetermined amount of said solvent solution, sedimentation of the DNA and removal of any phenol and salt contamination from the DNA.
18. The method of claim 17 wherein said water-insoluble organic solvent added to said homogenate is chloroform.
19. The method of claim 17 wherein the lower alcohol added to the aqueous phase is isopropanol.
20. The method of claim 17 wherein the lower alcohol added to the organic phase is isopropanol.
21. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:
- (a) homogenizing a tissue sample in the solvent solution of claim 1 to form a homogenate;
- (b) adding a water-insoluble organic solvent to said homogenate and sedimenting to form a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA;
- (c) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;
- (d) extracting the organic phase and interphase with water;
- (e) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and
- (f) precipitating DNA from the interphase by the addition of CsCl, sodium citrate solution and a lower alcohol thereto and recovering the precipitated DNA by sedimentation.
22. The method of claim 21 wherein said water-insoluble organic solvent added to said homogenate is chloroform.
23. The method of claim 21 wherein the lower alcohol added to the aqueous phase is isopropanol.
24. The method of claim 21 wherein the lower alcohol added to the organic phase is isopropanol.
25. The method of claim 21 wherein the lower alcohol added to the interphase is ethanol.
26. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:
- (a) homogenizing a tissue sample in the solvent solution of claim 1 to form a homogenate;
- (b) sedimenting substantially pure, undegraded DNA from said homogenate, washing the sedimented DNA with an amount of said solvent solution, and removing any phenol and salt contamination from the DNA;
- (c) adding a water-insoluble organic solvent to the residual homogenate subsequent to said DNA sedimenting step, and thereafter sedimenting to form a mixture having an aqueous phase containing substantially pure, undegraded RNA and an organic phase containing proteins;
- (d) precipitating RNA from the aqueous phase by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;
- (e) precipitating proteins from the organic phase by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation.
27. The method of claim 26 wherein said water-insoluble organic solvent added to said residual homogenate is chloroform.
28. The method of claim 26 wherein said lower alcohol added to the aqueous phase is isopropanol.
29. The method of claim 26 wherein said lower alcohol added to the organic phase is isopropanol.
30. A method of isolating substantially pure RNA, DNA and proteins from biological tissue, comprising the steps of:
- (a) precipitating RNA from an aqueous phase obtained from a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA, said mixture formed by adding a water-insoluble organic solvent to a homogenate and sedimenting, said homogenate formed by homogenizing a tissue sample in a solvent solution comprising effective amounts of phenol and a guanidinium compound for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue, by the addition of a lower alcohol thereto and recovering the precipitated RNA by sedimentation;
- (b) precipitating proteins from an organic phase obtained from a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA, said mixture formed by adding a water-insoluble organic solvent to a homogenate and sedimenting, said homogenate formed by homogenizing a tissue sample in a solvent solution comprising effective amounts of phenol and a guanidinium compound for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue, by the addition of a lower alcohol thereto and recovering the precipitated proteins by sedimentation; and
- (c) recovering DNA from an interphase obtained from a mixture consisting of an aqueous phase containing substantially pure, undegraded RNA, an organic phase containing proteins, and an interphase containing substantially pure, undegraded DNA, said mixture formed by adding a water-insoluble organic solvent to a homogenate and sedimenting, said homogenate formed by homogenizing a tissue sample in a solvent solution comprising effective amounts of phenol and a guanidinium compound for extracting substantially pure and undegraded RNA, substantially pure and undegraded DNA, and proteins from biological tissue, by washing the interphase with a predetermined amount of said solvent solution, sedimentation of the DNA and removal of any phenol and salt contamination from the DNA.