Methods and compositions for analysis of microRNA
The invention provides methods and systems for detecting and measuring microRNAs.
1 . A method for quantitating microRNA in a sample comprising
contacting a template nucleic acid with a microRNA and allowing the template nucleic acid to bind to the microRNA thereby creating a 5′ template overhang,
polymerizing a nucleic acid tail onto the microRNA wherein the nucleic acid tail is complementary to the 5′ template overhang and thereby creating a tailed microRNA,
separating the template nucleic acid from the tailed microRNA,
contacting a first and a second sequence specific probe with the tailed microRNA and allowing the first and second sequence specific probes to bind to the tailed microRNA wherein the first and second sequence specific probes are complementary to the microRNA or the nucleic acid tail,
contacting the tailed microRNA with a solid support conjugated to a nucleic acid complementary to the nucleic acid tail and allowing the tailed microRNA to bind to the solid support at a defined location, and
detecting the level of binding of the tailed microRNA to the solid support based on the presence of the first and second sequence specific probes at the defined location.
2 . The method of claim 1 , wherein the first and second sequence specific probes are conjugated to first and second detectable labels.
3 . The method of claim 2 , wherein the first and second detectable labels are first and second fluorophores.
4 . The method of claim 3 , wherein the first fluorophore is distinct from the second fluorophore.
5 . The method of claim 1 , wherein the template nucleic acid is about 50% longer than the microRNA.
6 . The method of claim 1 , wherein the 5′ template overhang is at least 10 bases in length.
7 . The method of claim 1 , wherein the tailed microRNA is contacted with the first and second sequence specific probes prior to contact with the solid support.
8 . The method of claim 1 , wherein the tailed microRNA is contacted with the first and second sequence specific probes after contact with and binding to the solid support
9 . The method of claim 1 , wherein the microRNA is less than 25 nucleotides in length.
10 . The method of claim 1 , wherein the template nucleic acid is a DNA.
11 . The method of claim 1 , wherein the first and second sequence specific probes are comprised of LNA or are LNA/DNA chimerae.
12 . The method of claim 1 , wherein the solid support is a silica chip.
13 . The method of claim 1 , further comprising quantitating a plurality of microRNA.
14 . The method of claim 1 , wherein the defined location on the solid support has a plurality of nucleic acids conjugated to it.
15 . The method of claim 1 , wherein the nucleic acid tail is polymerized by a primer extension reaction.
16 . The method of claim 15 , wherein the primer extension reaction comprises a thermophilic exopolymerase.
17 . The method of claim 1 , wherein the nucleic acid tail is fluorescent.
18 . The method of claim 1 , wherein the nucleic acid complementary to the nucleic acid tail is comprised of an LNA or is an LNA/DNA chimera.
19 . The method of claim 1 , wherein the nucleic acid complementary to the nucleic acid tail is tethered to the solid support via a 3′ ethylene glycol scaffold.