Methods and compositions for isolating small RNA molecules
First Claim
Patent Images
1. A method for isolating small RNA molecules from cells comprising:
- a) lysing the cells with a lysing solution to produce a lysate;
b) adding an alcohol solution to the lysate;
c) applying the lysate to a solid support;
d) eluting small RNA molecules from the solid support; and
, e) using or characterizing the small RNA molecules.
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Abstract
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.
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Citations
53 Claims
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1. A method for isolating small RNA molecules from cells comprising:
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a) lysing the cells with a lysing solution to produce a lysate;
b) adding an alcohol solution to the lysate;
c) applying the lysate to a solid support;
d) eluting small RNA molecules from the solid support; and
,e) using or characterizing the small RNA molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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42. A method for isolating miRNA or siRNA from a sample comprising:
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a) obtaining a sample having miRNA or siRNA;
b) adding an alcohol solution to the sample;
c) adding an extraction solution to the sample;
c) applying the sample to a mineral or polymer support; and
d) eluting the siRNA or miRNA from the mineral or polymer support with an ionic solution. - View Dependent Claims (43, 44, 45)
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46. A method for isolating miRNA molecules from a sample comprising:
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a) adding an alcohol solution to the sample;
b) applying the sample to a mineral or polymer support;
c) eluting miRNA molecules from the support with an ionic solution; and
d) using or characterizing the miRNA molecules. - View Dependent Claims (47)
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48. A method for isolating small RNA molecules from a sample comprising:
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a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced;
b) extracting small RNA molecules from the lysate with an extraction solution comprising phenol;
c) adding to the lysate an alcohol solution for form a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%;
d) applying the lysate/alcohol mixture to a mineral or polymer support;
e) eluting the small RNA molecules from the mineral or polymer support with an ionic solution;
f) capturing the small RNA molecules; and
g) using the isolated small RNA molecules.
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49. A kit for isolating small RNA molecules comprising:
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a) acid phenol-chloroform;
b) lysis/binding buffer;
c) homogenate additive;
d) wash solution;
e) elution solution; and
f) filter cartridges
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50. A method for isolating small RNA molecules from a sample comprising:
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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. - View Dependent Claims (51, 52, 53)
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Specification