METHODS OF DETECTING miRNA

Abstract

Described are non-invasive methods of detecting in vivo cell death by measuring levels of ubiquitous and tissue specific miRNA. The method can be applied for detection of pathologies caused or accompanied by cell death, as well as for diagnosis of infectious disease, cytotoxic effects induced by different chemical or physical factors, and the presence of specific fetal abnormalities.

1 . A method of detecting at least one cell-free miRNA released from at least one cell in a subject, the method comprising:
 a) separating a soluble fraction of a sample of urine or blood obtained from a subject; and   b) detecting and quantitating at least one cell-free miRNA in the soluble fraction with at least one oligonucleotide primer or probe that is substantially complementary to a part of said at least one cell-free miRNA,   wherein the at least one cell-free miRNA is indicative of in vivo cell death associated with a disease or disorder.   
     
     
         2 . The method of  claim 1 , wherein said disorder is a brain stroke. 
     
     
         3 . The method of  claim 1 , wherein said disorder is Alzheimer's disease. 
     
     
         4 . The method of  claim 1 , wherein said disorder is Parkinson's disease. 
     
     
         5 . The method of  claim 1 , wherein said at least one cell-free miRNA is associated with fetal pathology. 
     
     
         6 . The method of  claim 5 , wherein said fetal pathology is Down syndrome. 
     
     
         7 . The method of  claim 1 , wherein said disorder is a pathogen infection, such as a virus infection. 
     
     
         8 . The method of  claim 1 , wherein said detecting and quantitating diagnoses said disease or disorder. 
     
     
         9 . The method of  claim 1 , wherein said detecting and quantitating is repeated over time to monitor the progression or regression of said disease or disorder. 
     
     
         10 . A method of detecting at least one cell-free miRNA released from at least one cell in a subject, the method comprising:
 a) separating a soluble fraction of a sample of urine or blood obtained from a subject; and   b) detecting and quantitating at least one cell-free miRNA in the soluble fraction with at least one oligonucleotide primer or probe that is substantially complementary to a part of said at least one cell-free miRNA,   wherein the at least one cell-free miRNA is indicative of in vivo cell death associated with acute pathology, chronic pathology, or the cytotoxic effect of physical or chemical agents.   
     
     
         11 . The method of  claim 10 , wherein the acute pathology is selected from acute myocardial infarction associated with death of cardiomyocytes, hepatitis or liver cirrhosis associated with hepatocyte death caused by a viral or other infection or by action of toxic agents, acute pancreatitis associated with death of different pancreatic cells, rejection of a transplanted organ associated with excessive cell death in the transplanted organ, traumatic damage of various organs, acute infections, or tuberculosis associated with cell death in lungs and/or other infected organs. 
     
     
         12 . The method of  claim 10 , wherein the chronic pathology is selected from frontotemporal dementia, chronic heart failure associated with the death of cardiomyocytes, emphysema associated with death of lung cells, diabetes type 1 associated with the death of pancreatic beta cells, glomerulonephritis associated with the death of kidney cells, precancerous conditions associated with the apoptotic death of actively proliferating precancerous cells, cancers associated with massive necrotic cell death due to insufficient blood supply, and cell death in chronically infected organs or tissues. 
     
     
         13 . The method of  claim 10 , wherein the cytotoxic effect of physical or chemical agents is selected from radiation associated with doses that kill bone marrow cells, doses that lead to the death of epithelial cells of gastrointestinal system, and doses that kill brain neurons, and chemical cytotoxicity associated with cell death in different organ and tissues induced by natural or synthetic toxic compounds. 
     
     
         14 . A method of determining the prognosis the outcome of a disease or disorder, the method comprising:
 a) separating a soluble fraction of a sample of urine or blood obtained from a subject; and   b) detecting and quantitating at least one cell-free miRNA in the soluble fraction with at least one oligonucleotide primer or probe that is substantially complementary to a part of said at least one cell-free miRNA,   wherein the at least one cell-free miRNA is indicative of the outcome of a disease or disorder.   
     
     
         15 . The method of  claim 14 , wherein a change in the level of the at least one cell-free miRNA over time is indicative of the progression or regression of a disease or disorder, or is indicative of the success of therapeutic or surgical intervention, or monitors progress of a treatment in the subject. 
     
     
         16 . The method of  claim 1 , wherein said sample is urine. 
     
     
         17 . The method of  claim 1 , wherein said soluble fraction is serum. 
     
     
         18 . The method of  claim 1 , wherein said soluble fraction is plasma.