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 molecules of from 10 to 100 nucleotides in length from cells comprising:
a) lysing the cells with a lysing solution to produce a lysate;
b) adding an alcohol solution to the lysate; wherein the alcohol concentration in the lysate is at least about 55% to about 70%;
c) applying the lysate to a solid support;
d) eluting small RNA molecules of from 10 to 100 nucleotides in length from the solid support wherein the number of small molecules is enriched at least 3 fold when compared to the total number of RNA molecules; and,
e) using or characterizing the small RNA molecules.
2. The method of claim 1 , wherein the lysing solution comprises a chaotropic agent or detergent.
3. The method of claim 2 , wherein the lysing solution comprises a chaotropic agent.
4. The method of claim 1 , further comprising extracting small RNA molecules from the lysate with an extraction solution comprising an organic solvent prior to applying the lysate to the solid support.
5. The method of claim 1 , wherein the small RNA molecules are eluted from the solid support with a low-ionic-strength solution.
6. The method of claim 5 , wherein the ionic solution comprises up to 10 mM salt.
7. The method of claim 1 , wherein the solid support is a mineral support or polymer support.
8. The method of claim 7 , wherein the mineral support or polymer support is a column comprising silica.
9. The method of claim 8 , wherein the silica is glass fiber.
10. The method of claim 1 , wherein the RNA molecules have from 10 to 70 nucleotides.
11. The method of claim 10 , wherein the RNA molecules have from 10 to 30 nucleotides.
12. A method for isolating miRNA or siRNA from a sample comprising:
a) obtaining a sample having miRNA or siRNA;
b) adding an alcohol solution to the sample to produce a sample-alcohol solution wherein the sample-alcohol solution comprises an alcohol concentration of at least about 55% to about 70%;
c) adding an extraction solution to the sample-alcohol solution;
d) applying the sample to a mineral or polymer support; and
e) eluting the miRNA or siRNA from the mineral or polymer support with an ionic solution wherein the number of miRNA or siRNA molecules is enriched at least 3 fold when compared to the total number of RNA molecules.
13. The method of claim 12 , wherein the sample is a cell lysate.
14. The method of claim 13 , wherein the cell lysate is produced by adding a lysing solution comprising a chaotropic agent or detergent to cells having miRNA or siRNA.
15. A method for isolating miRNA molecules from a sample comprising:
a) adding an alcohol solution to the sample to produce a sample-alcohol solution wherein the sample-alcohol solution comprises an alcohol concentration of at least about 55% to about 70%;
b) applying the sample-alcohol solution to a mineral or polymer support; and
c) eluting the miRNA from the mineral or polymer support with an ionic solution wherein the number of miRNA molecules is enriched at least 3 fold when compared to the total number of RNA molecules; and
d) using or characterizing the miRNA molecules.
16. The method of claim 15 , wherein the sample is a cell lysate.