Compositions and methods for using a solid support to purify RNA
Reagents, methods and kits for the purification of RNA from biological materials are provided.
1. A method for purifying RNA from biological material comprising RNA, comprising:
(a) mixing said biological material with an RNA Lysing Solution, wherein the RNA Lysing Solution
(i) is buffered at a pH of greater than 7,
(ii) comprises an amphiphillic reagent and an RNA complexing salt, wherein the RNA-complexing salt is an alkali-metal salt present at a concentration greater than 4 M, and
(iii) is free of a strong chaotropic substance;
(b) lysing said biological material with said RNA Lysing Solution to form a lysate comprising RNA and non-nucleic acid biological matter;
(c) contacting said lysate to an immobilized non-silica solid support, wherein said RNA in said lysate preferentially binds to said solid support;
(d) washing said solid support with an RNA wash solution to remove non-nucleic acid biological matter; and
(e) preferentially eluting the bound RNA from said solid support with an RNA elution solution to obtain the RNA.
2. The method of claim 1 , wherein the biological material is selected from the group consisting of crude and partially purified mixtures of nucleic acids.
3. The method of claim 1 , wherein the biological material is selected from the group consisting of plant cells, mycoplasma, protozoa, bacteria, fungi, viruses, yeasts, rickettsia and homogenates thereof.
4. The method of claim 1 , wherein the biological material is selected from the group consisting of whole blood, bone marrow, blood spots, blood serum, blood plasma, buffy coat preparations, saliva, cerebrospinal fluid, and solid animal tissues.
5. The method of claim 1 , wherein the biological material is selected from the group consisting of feces, urine, tears, and sweat.
6. The method of claim 1 , wherein the biological material is selected from the group consisting of environmental samples taken from air, water, sediment and soil.
7. The method of claim 1 , wherein the non-silica solid support comprises components selected from a group consisting of cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, polyvinylidene fluoride, and combinations thereof.
8. The method of claim 1 , wherein the non-silica solid support comprises a polyester.
9. The method of claim 1 , wherein the immobilized non-silica solid support comprises combinations of polyesters.
10. The method of claim 1 , wherein the solid support is contained in a vessel.
11. The method of claim 1 , wherein the RNA Lysing Solution is free of guanidinium salts and urea.
12. The method of claim 1 , wherein the RNA is selected from the group consisting of messenger RNA, transfer RNA, ribosomal RNA and viral RNA, and combinations thereof.
13. The method of claim 1 , wherein the alkali-metal salt is chosen from the group consisting of sodium, potassium, lithium, cesium, and rubidium salts.
14. The method of claim 13 , wherein the alkali-metal salt is a lithium salt.
15. The method of claim 14 , wherein the alkali-metal salt is lithium chloride.
16. The method of claim 1 , wherein the alkali-metal salt is present at a concentration of between 4-10 M.
17. The method of claim 1 , wherein the amphiphillic reagent is a detergent.
18. The method of claim 17 , wherein the detergent is a non-ionic detergent.
19. The method of claim 18 , wherein the nonionic detergent is selected from the group consisting of tweens, tritons, nomodets, and tergitols.
20. The method of claim 1 , wherein the RNA Lysing Solution comprises a chelating agent.
21. The method of claim 20 , wherein the chelating agent is selected from the group consisting of EDTA and CDTA.
22. A method for purifying RNA from biological material, comprising:
(a) contacting a biological material containing RNA with a solid support pre-treated with an RNA Lysing Solution to release RNA and non-nucleic acid biological matter and cause the released RNA to preferentially bind to said solid support, wherein the RNA Lysing Solution
(i) is buffered at a pH of greater than 7,
(ii) is bound to the solid support,
(iii) comprises an amphiphillic reagent and an RNA-complexing salt, wherein the RNA-complexing salt is an alkali-metal salt present at a concentration greater than 4 M, and
(iv) is free of a strong chaotropic substance;
(b) washing said solid support with an RNA wash solution to remove the non-nucleic acid biological materials; and
(c) preferentially eluting the bound RNA from said solid support with an RNA elution solution to obtain the RNA.
23. The method of claim 1 , wherein the RNA that is purified is substantially undegraded RNA.
24. The method of claim 10 , wherein the vessel is a centrifuge tube, spin tube, syringe, cartridge, chamber, multiple-well plate or test tube.
25. The method of claim 22 , wherein the biological material is selected from the group consisting of crude and partially purified mixtures of nucleic acids.
26. The method of claim 22 , wherein the biological material is selected from the group consisting of plant cells, mycoplasma, protozoa, bacteria, fungi, viruses, yeasts, rickettsia and homogenates thereof.
27. The method of claim 22 , wherein the biological material is selected from the group consisting of whole blood, bone marrow, blood spots, blood serum, blood plasma, buffy coat preparations, saliva, cerebrospinal fluid, and solid animal tissues.
28. The method of claim 22 , wherein the biological material is selected from the group consisting of feces, urine, tears, and sweat.
29. The method of claim 22 , wherein the biological material is selected from the group consisting of environmental samples taken from air, water, sediment and soil.
30. The method of claim 22 , wherein the solid support is a non-silica solid support.
31. The method of claim 30 , wherein the non-silica solid support comprises components selected from the group consisting of cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, polyvinylidene fluoride, and combinations thereof.
32. The method of claim 30 , wherein the non-silica solid support comprises a polyester.
33. The method of claim 22 , wherein the immobilized non-silica solid support comprises combinations of polyesters.
34. The method of claim 22 , wherein the solid support is contained in a vessel.
35. The method of claim 22 , wherein the RNA Lysing Solution is free of guanidinium salts and urea.
36. The method of claim 22 , wherein the RNA is selected from the group consisting of messenger RNA, transfer RNA, ribosomal RNA and viral RNA, and combinations thereof.
37. The method of claim 22 , wherein the alkali-metal salt is chosen from the group consisting of sodium, potassium, lithium, cesium, and rubidium salts.
38. The method of claim 37 , wherein the alkali-metal salt is a lithium salt.
39. The method of claim 38 , wherein the alkali-metal salt is lithium chloride.
40. The method of claim 22 , wherein the alkali-metal salt is present at a concentration of between 4-10 M.
41. The method of claim 22 , wherein the amphiphillic reagent is a detergent.
42. The method of claim 22 , wherein the detergent is a non-ionic detergent.
43. The method of claim 42 , wherein the non-ionic detergent is selected from the group consisting of tweens, tritons, nomodets, and tergitols.
44. The method of claim 22 , wherein the RNA Lysing Solution comprises a chelating agent.
45. The method of claim 44 , wherein the chelating agent is selected from the group consisting of EDTA and CDTA.
46. The method of claim 22 , wherein the RNA that is purified is substantially undegraded RNA.