Method for isolating total RNA from cells
View Patent ↗Provided herein are methods for isolating cellular ribonucleic acid (RNA) from cells. The method includes suspending cells in an extraction solution comprising formamide; incubating the cells and formamide mixture; and pelleting cell debris, DNA, and protein to form an RNA-containing supernatant. Also provided herein are kits for isolating RNA and solutions for extracting RNA from a cell.
1. A method for isolating cellular ribonucleic acid (RNA), comprising:
(a) suspending cells in an extraction solution comprising formamide in an amount of at least 40% by volume of the extraction solution;
(b) incubating the cells and formamide mixture of step (a); and
(c) pelleting cell debris, DNA, and protein to form an RNA-containing supernatant.
2. The method of claim 1 , further comprising adding zirconium beads prior to incubating the mixture.
3. The method of claim 1 , further comprising pelleting the cells prior to suspending the cells in the extraction solution.
4. The method of claim 1 , wherein the extraction solution further comprises a reducing agent, an anionic detergent, a chelating agent, or a mixture thereof.
5. The method of claim 4 , wherein the reducing agent comprises β-mercaptoethanol or dithiothreitol.
6. The method of claim 5 , wherein the β-mercaptoethanol is present in the extraction solution in an amount of less than 5% by volume of the extraction solution.
7. The method of claim 5 , wherein the β-mercaptoethanol is present in the extraction solution in an amount of less than 2% by volume of the extraction solution.
8. The method of claim 5 , wherein the dithiothreitol is present in the extraction solution at a concentration of from 50 μM to 1 M.
9. The method of claim 4 , wherein the anionic detergent comprises sodium dodecyl sulfate.
10. The method of claim 4 , wherein the anionic detergent is present in the extraction solution in an amount of less than 5% by weight/volume of the extraction solution.
11. The method of claim 4 , wherein the anionic detergent is present in the extraction solution in an amount of less than 0.05% by weight/volume of the extraction solution.
12. The method of claim 4 , wherein the chelating agent comprises EDTA.
13. The method of claim 4 , wherein the chelating agent is present in the extraction solution at a concentration of from 0.5 mM to 50 mM.
14. The method of claim 4 , wherein the chelating agent is present in the extraction solution at a concentration of 18 mM.
15. The method of claim 1 , wherein the extraction solution is substantially free from quaternary amine surfactants.
16. The method of claim 1 , wherein the incubating step is performed at a temperature of from 10° C. to 140° C.
17. The method of claim 1 , wherein the incubating step is performed at a temperature of 95° C.