Methods and kits for improving global gene expression analysis of human urine derived rna

Abstract

Disclosed are methods and kits for improving global gene expression analysis for a population of RNA molecules derived from a human urine sample. In an embodiment, the method comprises the step of selectively depleting miR-10a-5p fragments from the population of RNA molecules or selectively blocking miR-10a-5p fragments within the RNA population. The miR-10a-5p depleted or miR-10a-5p blocked population of RNA can be used in a variety of global gene expression analysis protocols, including next generation sequencing. In a further embodiment, the method comprises selectively depleting or blocking miR-10b-5p fragments within the RNA population. The miR-10a-5p and/or miR-10b-5p depleted or blocked populations of RNA can also be used in global gene expression analysis protocols, including next generation sequencing. The kit comprises oligonucleotide probes comprising a nucleotide sequence that is the complement to a nucleotide sequence of the miR-10a-5p and/or oligonucleotide probes comprising a nucleotide sequence that is the complement to a nucleotide sequence of miR-10b-5p.

1 . A method of improving global gene expression analysis for a population of RNA molecules derived from human urine, the method comprising the step of blocking miR-10a-5p fragments and/or miR-10b-5p fragments in the population of RNA molecules. 
     
     
         2 . The method of  claim 1 , wherein the step of blocking the miR-10a-5p fragments and/or miR-10b-5p fragments in the population of RNA molecules comprises:
 adding miR-10a-5p specific oligonucleotide probes and/or miR-10b-5p specific oligonucleotide probes to a sample containing the population of RNA molecules, wherein each miR-10a-5p specific oligonucleotide probe comprises a nucleotide sequence that is the complement to a nucleotide sequence of miR-10a-5p and each miR-10b-5p specific oligonucleotide probe comprises a nucleotide sequence that is the complement a nucleotide sequence of miR-10b-5p; and   forming a complex between one or more miR-10a-5p fragments and a miR-10a-5p specific oligonucleotide probe and/or forming a complex between one or more miR-10b-5p fragments and a miR-10b-5p specific oligonucleotide probe to provide a miR-10a-5p and/or miR-10b-5p blocked sample.   
     
     
         3 . The method of  claim 2 , wherein the 5′end, the 3′end or both ends of each miR-10a-5p specific oligonucleotide probes and/or miR-10b-5p specific oligonucleotide probes is modified to prevent ligation and wherein the modification is a 5′ biotin modification, a 3′ biotin modification, a 5′ dioxigenin modification, a 3′ dioxigenin modification, a 5′ dinitrophenol modification, 5′ dideoxy nucleotide modification, a 3′ dideoxy nucleotide modification or a combination thereof. 
     
     
         4 . The method of  claim 2 , wherein the nucleotide sequence of miR-10a-5p has at least 90% identity to the nucleotide sequence of SEQ ID NO: 1 and wherein the nucleotide sequence of miR-10b-5p has at least 90% identity to the nucleotide SEQ ID NO: 3. 
     
     
         5 . The method of  claim 2 , wherein the miR-10a-5p specific oligonucleotide probe has at least 90% identity to the nucleotide sequence of SEQ ID NO: 2 and the miR-10b-5p specific oligonucleotide probe has at least 90% identity to the nucleotide sequence of SEQ ID NO: 4. 
     
     
         6 . The method of  claim 2 , wherein the global gene expression analysis is next generation sequencing and wherein the method further comprises the steps of:
 preparing a library using the miR-10a-5p and/or miR-10b-5p blocked sample; and   sequencing the library.