Automated rna detection using labeled 2'-o-methyl rna oligonucleotide probes and signal amplification systems
AssigneeVentana Medical Systems, Inc.
InventorsWilliam Day, Michael Farrell, Zeyu Jiang, Anne Pedata
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Abstract
Disclosed herein are methods and compositions for detecting differential expression of certain miRNAs in cancer cells or their surrounding normal tissues in the tumor microenvironment. The disclosure describes an automated, highly sensitive and specific method for detection of any cellular RNA molecule, including microRNA, messenger RNA and non-coding RNA. The technology includes probe design as well as probe use in an automated fashion for detection of RNA molecules in formalin-fixed paraffin-embedded tissue (FFPET) samples.
Claims22
A system for bright field in situ hybridization comprising:
- a probe set comprising X unique 2′-O-methyl RNA probes specific to a target RNA, wherein X≥2, the probes target X distinct portions within the target RNA, wherein each 2′-O-methyl RNA probe is conjugated with at least one detectable moiety, the detectable moiety being adapted to bind a reactive chromogen conjugate system for signal amplification.
14-24. (canceled)
A method of in situ hybridization comprising:
- contacting a sample with a probe set specific for a target RNA under conditions sufficient that the probe set hybridizes to the target RNA in the sample, wherein the probe set comprises X unique 2′-O-methyl RNA probes, wherein X≥2, the 2′-O-methyl RNA probes target X distinct portions within the target RNA, and wherein each 2′-O-methyl RNA probe is conjugated with at least one detectable moiety;
- contacting the sample with a first anti-detectable moiety antibody conjugated with a first enzyme, the first anti-detectable moiety antibody being specific for the X unique RNA probes;
- contacting the sample with a reactive chromogen conjugate, wherein the first enzyme of the first anti-detectable moiety antibody binds the reactive chromogen conjugate to the first anti-detectable moiety antibody and surrounding tissue;
- and
- contacting the sample with a second antibody conjugated with a second enzyme, the second antibody is specific for the reactive chromogen conjugate, wherein the second enzyme catalyzes visibility of the chromogen, and wherein visibility of the chromogen is indicative of the target RNA.
A method of in situ hybridization comprising:
- contacting a sample with a 2′-O-methyl RNA probe specific for a target RNA under conditions sufficient that the 2′-O-methyl RNA probe hybridizes to the target RNA in the sample, wherein the 2′-O-methyl RNA probe is conjugated with at least one detectable moiety disposed at either the 5′ end or the 3′ end, and wherein the 2′-O-methyl probe is between 15 to 30 nucleotides in length;
- contacting the sample with a first anti-detectable moiety antibody conjugated with a first enzyme, wherein the first anti-detectable moiety antibody is specific for the 2′-O-methyl RNA probe;
- contacting the sample with a reactive chromogen conjugate, wherein the first enzyme of the first anti-detectable moiety antibody binds the reactive chromogen conjugate to the first anti-detectable moiety antibody;
- and
- contacting the sample with a second antibody conjugated with a second enzyme, wherein the second antibody is specific for the reactive chromogen conjugate, and wherein the second enzyme catalyzes visibility of the chromogen, wherein visibility of the chromogen is indicative of the target RNA.
Dependencies are inferred from the claim text.