Methods and compositions for isolating small RNA
The present invention concerns the use of methods and compositions for the isolation of small RNA molecules (100 nucleotides or fewer), such as microRNA and siRNA molecules. Such molecules are routinely lost in commonly used isolation procedures and therefore the present invention allows for a much higher level of enrichment or isolation of these small RNA molecules.
1. A method for isolating small RNA molecules having at most 100 nucleotides or fewer from a sample comprising:
a) lysing cells in a lysing solution to produce a lysate; wherein the lysing solution comprises a chaotropic agent;
b) adding to the lysate an ethanol solution to form a lysate/ethanol mixture;
c) applying the lysate/ethanol mixture to a first solid support and collecting flow-through lysate/ethanol mixture;
d) adding to the flow-through lysate/ethanol mixture of step c) an ethanol solution;
e) applying the lysate/ethanol mixture of step d) to a second solid support; and
f) eluting small RNA molecules from the solid support, wherein the lysate/ethanol mixture applied to the first solid support is between about 20% to about 35% ethanol and wherein the lysate/ethanol mixture applied to the second solid support is between about 35% to about 70% ethanol.
2. The method of claim 1 , wherein the lysing solution comprises a detergent.
3. The method of claim 2 , wherein the concentration of the detergent in the lysing solution is about 0.1% to about 2%.
4. The method of Claim 2 wherein the chaotropic agent is a guanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.
5. The method of Claim 2 wherein the chaotropic agent is a guanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.
6. The method of Claim 3 wherein the chaotropic agent is a guanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.
7. The method of Claim 1 , wherein the lysate is extracted with phenol and/or chloroform.
8. The method of Claim 2 , wherein the lysate is extracted with phenol and/or chloroform.
9. The method of Claim 4 , wherein the lysate is extracted with phenol and/or chloroform.
10. The method of Claim 1 , wherein the lysing solution comprises a buffer in a concentration of about 10 mM to about 300 mM.
11. The method of Claim 7 , wherein the lysing solution comprises a buffer in a concentration of about 10 mM to about 300 mM.
12. The method of Claim 1 , further comprising washing the solid support with a first wash solution after applying the lysate to the solid support and optionally washing the solid support with a second wash solution after washing with the first wash solution.
13. The method of claim 12 , wherein the first wash solution comprises a chaotropic agent wherein the chaotropic agent is a guanidinium salt and the first wash solution further comprises alcohol.
14. The method of claim 13 , wherein the second wash solution comprises alcohol.
15. The method of Claim 1 , wherein the small RNA molecules are eluted from the solid support at a temperature of about 60° C. to about 100° C.
16. The method of Claim 4 , wherein the small RNA molecules are eluted from the solid support with a low-ionic-strength solution.
17. The method of Claim 1 , wherein the solid support comprises silica.
18. The method of Claim 1 , wherein the first and the second solid support are made of the same material.
19. The method of Claim 17 , wherein the solid support is glass fiber filter or column.