Methods and compositions for isolating small RNA molecules
View Patent ↗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 RNA including small RNA molecules of from 10 to 100 nucleotides in length from cells comprising:
a) lysing the cells with a lysing solution to produce a lysate wherein the lysing solution comprises guanidinium;
b) extracting the lysate with an extracting solution comprising a non-alcohol organic solvent to form an aqueous phase and an organic phase;
c) adding an alcohol solution to the aqueous phase to form a mixture of 50% to 60% alcohol;
d) applying the mixture to a solid support;
e) washing the solid support with a wash solution; and
f) eluting RNA including small RNA molecules from the solid support.
2. The method of claim 1 , wherein the small RNA molecules include miRNA, siRNA, snRNA, snoRNA, and/or tRNA molecules.
3. The method of claim 2 , wherein the small RNA molecules are miRNA molecules.
4. The method of claim 1 , wherein at least 20% of the small RNA molecules from the cells are isolated.
5. The method of claim 4 , wherein at least 50% of the small RNA molecules from the cells are isolated.
6. The method of claim 1 , wherein the wash solution comprises a chaotropic agent.
7. The method of claim 6 , wherein the chaotropic agent is guanidinium and the wash solution further comprises alcohol.
8. The method of claim 1 , further comprising washing the solid support with a second wash solution.
9. The method of claim 8 , wherein the second wash solution comprises alcohol.
10. 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.
11. The method of claim 1 , wherein the small RNA molecules are eluted from the solid support with a low-ionic-strength solution.
12. The method of claim 11 , wherein the ionic solution comprises up to 10 mM salt.
13. The method of claim 1 , wherein the solid support is a mineral support or polymer support.
14. The method of claim 13 , wherein the mineral support or polymer support is a column comprising silica.
15. The method of claim 13 , wherein the mineral or polymer support is a set of beads made of an absorptive polymer.
16. The method of claim 15 , wherein the set of beads are collected by centrifugation, filtration, or magnetic capture.
17. The method of claim 14 , wherein the silica is glass fiber.
18. The method of claim 1 , wherein, in step (c), adding the alcohol solution to the aqueous phase forms a mixture of 55% to 60% alcohol.