Methods and compositions involving MicroRNA
The present invention concerns methods and compositions for isolating, enriching, and/or labeling miRNA molecules and for preparing and using arrays or other detection techniques for miRNA analysis. Moreover, the present invention concerns methods and compositions for generating miRNA profiles and employing such profiles for therapeutic, diagnostic, and prognostic applications.
1. A method for multi-labeling miRNA in a sample comprising:
a) enriching miRNA with respect to total RNA in the sample;
b) forming a reaction mixture, under conditions that allows enzyme catalysis, comprising the miRNA with i) an enzyme that catalyzes the addition of di- or tri-phosphate nucleotides to the 3′ end of the miRNA; and ii) one or more labeled or unlabeled nucleotides, wherein tailed miRNA molecules are produced;
c) if unlabeled nucleotides are added to the miRNA, the method further comprising attaching a label to the tailed miRNA molecules,
wherein multi-labeled RNA are produced.
2. The method of claim 1 , wherein the enzyme is selected from the group consisting of poly(A) polymerase, terminal transferase, and polynucleotide phosphorylase.
3. The method of claim 2 , wherein the enzyme is poly(A) polymerase.
4. The method of claim 1 , wherein the nucleotides include one or more of uridine, adenosine, guanosine, or cytosine.
5. The method of claim 4 , wherein one or more of the nucleotides is a modified nucleotide.
6. The method of claim 5 , wherein the modified nucleotide is an amine-modified nucleotide.
7. The method of claim 6 , wherein the amine-modified nucleotide is selected from the group consisting of 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N 6 -(4-amino)butyl-ATP, N 6 -(6-amino)butyl-ATP, N 4 [2,2-oxy-bis-(ethylamine)]-CTP; N 6 -(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP.
8. The method of claim 1 , wherein the reaction mixture comprises labeled nucleotides.
9. The method of claim 1 wherein the label on the labeled nucleotides or the label attached to the unlabeled nucleotides is biotin, radioactivity, or a dye.
10. The method of claim 9 , wherein the dye is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 647, Alexa Fluor 594, BODIPY 630/650, BODIPY 650/665, BODIPY FL, BODIPY TMR, BODIPY TR, Cascade Blue, Cy-3, Cy-5, Fluorescein, Oregon Green 488, Rhodamine Green, Tetramethylrhodamine, and Texas Red.
11. The method of claim 5 , wherein the reaction mixture comprises modified and unmodified nucleotides.
12. The method of claim 6 , wherein the amine-modified nucleotides in the tailed miRNA of b) are unlabeled and a label is attached in c).
13. The method of claim 1 , wherein the reaction mixture further comprises a volume exclusion reagent.
14. The method of claim 1 , further comprising isolating RNA from the sample and then separating the miRNA from the other RNA.
15. The method of claim 14 , wherein the miRNA are separated from the other RNA by gel electrophoresis.
16. The method of claim 1 , further comprising:
d) isolating the multi-labeled miRNA molecules.