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 small RNA molecules from a sample comprising:
a) lysing cells in a lysing solution to produce a lysate;
b) extracting small RNA molecules from the lysate with an extraction solution comprising phenol;
c) adding to the lysate an alcohol solution to form a lysate/alcohol mixture;
d) applying the lysate/alcohol mixture to a first solid support;
e) collecting flow-through lysate/alcohol mixture;
f) adding to the flow-through lysate/alcohol mixture an alcohol solution;
g) applying the lysate/alcohol mixture to a second solid support; and
h) eluting small RNA molecules from the solid support with an ionic solution.
2. The method of claim 1 , wherein the lysate/alcohol mixture applied to the first solid support is between about 20% to about 35% alcohol.
3. The method of claim 1 , wherein the lysate/alcohol mixture applied to the second solid support is between about 35% to about 70% alcohol.
4. The method of claim 1 , further comprising using or characterizing the small RNA molecules.
5. A method for isolating small RNA molecules from a sample containing cells, the method comprising:
a) lysing the cells in a lysing solution to produce a lysate wherein the lysing solution comprises a chaotropic agent;
b) extracting RNA molecules from the lysate with an extraction solution comprising phenol to form an aqueous phase containing RNA molecules;
c) adding to the aqueous phase an alcohol solution to form a mixture of about 20% to about 35% alcohol;
d) applying the mixture of about 20% to about 35% alcohol to a first solid support wherein small RNA molecules flow through the solid support and large RNA molecules bind to the solid support;
e) collecting flow-through containing the small RNA molecules;
f) adding to the flow-through containing small RNA molecules an alcohol solution to an alcohol concentration of about 55% to about 70%;
g) applying the about 55% to about 70% alcohol solution containing small RNA molecules to a second solid support wherein small RNA molecules bind to the second solid support; and
h) eluting small RNA molecules from the second solid support
wherein small RNA molecules have 200 nucleotides or fewer, and large RNA molecules have greater than 200 nucleotides.