Methods for isolation, identification, and quantification of mirnas

Abstract

Method and compositions and kits for isolation, identification, and quantification of miRNAs and other small RNAs, including but not limited to, siRNAs, mRNAs, and snRNAs are disclosed. Methods of diagnosing a disease or its progression are also disclosed.

1 . A method for isolating, identifying, or quantifying an miRNA from a sample of interest, comprising:
 a) contacting the sample of interest with an anti-miRNA probe covalently attached to a bead;   b) incubating the sample of interest—bead mixture under suitable conditions to form hybridized complexes between the miRNA in the sample of interest and the anti-miRNA probes on the beads;   c) washing the beads under suitable conditions to remove the unbound sample material; and   d) isolating, identifying, or quantifying the miRNA bound to the anti-miRNA probes.   
     
     
         2 . The method according to  claim 1 , wherein the miRNA is isolated from the anti-miRNA probe beads before identifying or quantifying the miRNA. 
     
     
         3 . The method according to  claim 1 , wherein the identifying or quantifying the miRNA comprises using reverse transcription followed by real-time (RT) or quantitative polymerase chain reaction (q-PCR). 
     
     
         4 . The method according to  claim 1 , wherein the beads are magnetic beads. 
     
     
         5 . The method according to  claim 4 , wherein the beads are carboxylic acid beads. 
     
     
         6 . The method according to  claim 4 , wherein the beads are DNA beads. 
     
     
         7 . The method according to  claim 6 , wherein the beads are P1 DNA beads. 
     
     
         8 . The method according to  claim 1 , wherein the anti-miRNA probe is attached to the beads using chemical synthesis. 
     
     
         9 . The method according to  claim 1 , wherein the anti-miRNA probe is attached to the beads using enzymatic synthesis. 
     
     
         10 . The method according to  claim 9 , wherein the enzymatic synthesis comprises:
 a) taking an oligonucleotide that has a sequence complementary to an miRNA of interest (anti-miRNA probe);   b) adding a terminal didexoynucleotide to the 3′ end using a terminal transferase;   c) adding a 5′-phosphate group to the oligonucleotide using a kinase; and   d) ligating the oligonucleotide to DNA beads using a single-strand DNA ligase.   
     
     
         11 . The method according to  claim 1 , wherein only one species of anti-miRNA probe is attached to an individual bead. 
     
     
         12 . The method according to  claim 1 , wherein more than one species of anti-miRNA probes are attached to an individual bead. 
     
     
         13 . The method according to  claim 1 , wherein the sample of interest is a biological sample. 
     
     
         14 . The method according to  claim 13 , wherein the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, wound exudates, biopsies, autopsies, tissues, formalin-fixed, paraffin-embedded (PPFE) samples, and organs. 
     
     
         15 . A method according to  claim 14 , wherein the biological sample is a biological fluid. 
     
     
         16 . A method according to  claim 15 , wherein the biological fluid is blood. 
     
     
         17 . A method according to  claim 15 , wherein the biological fluid is serum. 
     
     
         18 . A method according to  claim 15 , wherein the biological fluid is saliva. 
     
     
         19 . A method of diagnosing a disease or disease progression, comprising:
 a) identifying a set of miRNA markers that are differentially regulated during a causation or progression of a disease;   b) isolating and measuring the levels of such miRNA markers from a suitable biological sample from a patient in need thereof, at different time points if necessary; and   c) diagnosing the disease or its progression over time in the patient.   
     
     
         20 . A kit for isolating, identifying, or quantifying an miRNA from a sample of interest, comprising:
 a) beads comprising anti-miRNA probe molecules covalently attached thereto;   b) a wash buffer; and   c) an elution buffer.