Method of RNA isolation from clinical samples
The disclosure provides methods for isolating nucleic acids from a biological fluid. In one aspect, the disclosure provides a method for isolating RNA. In another aspect, the disclosure provides a method for isolating DNA. In one aspect, the methods described herein utilize a protocol that combines a detergent-based initial denaturation, protease digestion, and organic extraction followed by column purification that maximizes RNA/DNA yield and preserves RNA/DNA integrity. In yet another aspect, the disclosure provides a kit for isolating RNA and/or DNA.
1. A method for isolating RNA from a biological fluid, said method comprising:
(a) contacting the biological fluid with a denaturing solution comprising at least one of reducing agent and detergent, wherein said denaturing solution is heated to about 50° C., about 55° C., about 60° C. or about 65° C. prior to contacting the biological fluid, to form a biological fluid mixture;
(b) contacting the biological fluid mixture with a protease to form a protease treated biological fluid mixture;
(c) contacting the protease treated biological fluid mixture with an organic extraction solution, forming a solution having an aqueous phase containing the RNA and an organic phase and wherein an interphase is absent or significantly reduced;
(d) binding the RNA to a silica based solid phase by contacting the aqueous phase with said silica based solid phase;
(e) contacting the silica based solid phase with a first wash solution comprising alcohol, chaotropic agent, and reducing agent; and
(f) eluting the RNA from the silica based solid phase comprising contacting the silica based solid phase with an aqueous solution to provide isolated RNA.
2. The method of claim 1 , wherein said biological fluid is selected from the group consisting of urine, serum, and plasma.
3. The method of claim 1 , wherein said biological fluid is homogenized tissue.
4. The method of claim 1 , wherein said protease is proteinase K.
5. The method of claim 1 , wherein said detergent is an anionic detergent.
6. The method of claim 5 , wherein said anionic detergent is SDS.
7. The method of claim 6 , wherein said SDS is present at a concentration of at least 2% (w/v), at least 3% (w/v), at least 4% (w/v), or at least 5% (w/v).
8. The method of claim 1 , wherein said chaotropic agent is selected from the group consisting of guanidine and urea.
9. The method of claim 1 , wherein said reducing agent is selected from the group consisting of DTT, BME, and TCEP.
10. The method of claim 1 , wherein said RNA is between 5-500 nucleotides, 5-250 nucleotides, 5-100 nucleotides, or 5-50 nucleotides.
11. The method of claim 1 , wherein said organic extraction solution further comprises at least one of chaotropic agent and phenol.
12. The method of claim 1 , wherein said organic extraction solution further comprises chloroform.
13. The method of claim 1 , wherein chloroform is contacted with the biological fluid after the biological fluid has contacted the organic extraction solution.
14. The method of claim 1 , wherein said reducing agent of step (e) is selected from the group consisting of DTT, BME, and TCEP.
15. The method of claim 14 , wherein said reducing agent is TCEP.
16. The method of claim 15 , wherein said TCEP has a concentration between 1 mM and 20 mM, between 1 mM and 10 mM, or between 1 and 5 mM.
17. The method of claim 8 , wherein said chaotropic agent is guanidine.
18. The method of claim 1 , wherein said alcohol of step (e) is selected from the group consisting of methanol, ethanol, butanol, and propanol.
19. The method of claim 1 , wherein said method further comprises at least one alcohol wash after step (e).
20. The method of claim 1 , wherein said aqueous solution of step (f) comprises at least one of water, buffer, and nucleic acid preservative.