MicroRNA detection
Provided herein are methods for detection of miRNA in a sample. In certain embodiments, the sample comprises RNA and is derived from a cell or tissue. The methods of detection employ a competitor molecule to allow for detection of an miRNA in the presence of an anti-miRNA oligonucleotide.
1. A method comprising:
contacting a cell with an anti-miRNA oligonucleotide;
isolating RNA from the cell to form a biological sample;
preparing a test sample comprising the biological sample;
adding at least one competitor molecule to the test sample, wherein the competitor molecule comprises an oligomeric compound comprising a nucleobase sequence that is at least 90% complementary to the nucleobase sequence of the anti-miRNA oligonucleotide; and
detecting free miRNA in the test sample, thereby detecting the miRNA amount in the biological sample.
2. The method of claim 1 , wherein the free miRNA is detected by separating free miRNA from the test sample by gel electrophoresis.
3. The method of claim 1 , wherein the anti-miRNA oligonucleotide has a melting temperature of less than 95° C.
4. The method of claim 1 , wherein the competitor molecule is added to the test sample in an amount from 50 to 150 pmol.
5. The method of claim 1 , wherein the competitor molecule is added in an amount from 5 to 15 pmol per 1 ug of RNA.
6. The method of claim 1 , wherein the competitor molecule is added in an amount not greater than 500 pmol per gel.
7. The method of claim 6 , wherein the oligomeric compound comprises a conjugate at least one terminus.
8. The method of claim 7 , wherein the conjugate is a solubility enhancing molecule.
9. The method of claim 8 , wherein the solubility enhancing molecule has the following Structure I:
10. The method of claim 1 , wherein the oligomeric compound comprises at least one high affinity modification.
11. The method of claim 1 , wherein the oligomeric compound comprises at least one peptide nucleic acid modification.
12. The method of claim 1 , wherein the oligomeric compound comprises a nucleobase sequence that is 100% complementary to the anti-miRNA oligonucleotide.
13. The method of claim 1 , wherein one or more nucleosides of the anti-miRNA oligonucleotide comprises at least one modified sugar.
14. The method of claim 1 , wherein each of a plurality of nucleosides of the anti-miRNA oligonucleotide comprises a modified sugar.
15. The method of claim 1 , wherein each nucleoside of the anti-miRNA oligonucleotide comprises a modified sugar.
16. The method of claim 1 , wherein the anti-miRNA oligonucleotide is conjugated to cholesterol.