Automated RNA detection using labeled 2′-O-methyl RNA oligonucleotide probes and signal amplification systems

Patent metadata

Field	Value
Publication number	US-10982269-B2
Application number	US-201615245126-A
Country	US
Kind code	B2
Filing date	Aug 23, 2016
Priority date	Feb 24, 2014
Publication date	Apr 20, 2021
Grant date	Apr 20, 2021

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.

First claim

The invention claimed is:

1. A system for bright field in situ detection of a microRNA (miRNA) target, the system comprising:
a target probe comprising a unique 2′-O-methyl RNA probe specific to the microRNA (miRNA) target, wherein the 2′-O-methyl RNA probe is conjugated with at least one detectable moiety disposed at either the 3′ end or the 5′ end of the target probe, wherein the target probe does not include locked nucleic acid (LNA) substitutions, and wherein the detectable moiety comprises a hapten; and
a reactive chromogen conjugate system effective for signal amplification, wherein the reactive chromogen conjugate system is adapted to bind to the detectable moiety of the target probe, and wherein the reactive chromogen conjugate system comprises a tyramide-hapten conjugate.

2. The system of claim 1 , wherein the 2′-O-methyl RNA probe comprises between 15 to 30 nucleotides.

3. The system of claim 1 , wherein the target probe comprises a first detectable moiety disposed at the 3′ end of the target probe, and a second detectable moiety disposed at the 5′ end of the target probe.

4. The system of claim 1 , wherein the hapten comprises dinitrophenol.

5. The system of claim 1 , further comprising a means of making the microRNA (miRNA) target visible.

6. The system of claim 5 , wherein the means of making the microRNA (miRNA) target visible comprises the step of contacting the target probes with the reactive chromogen conjugate system specific to the detectable moieties of the target probes, wherein the reactive chromogen conjugate system emits a color.

7. The system of claim 5 , further comprising a means of visualizing the microRNA (miRNA) target, wherein the detectable moieties are made visible by the reactive chromogen conjugate system, and wherein the visibility of the detectable moieties is indicative of the target microRNA.

8. The system of claim 7 , wherein the means of visualizing the microRNA (miRNA) target comprises a bright field microscope.

9. A method of bright field in situ hybridization comprising:
contacting a sample with an antigen retrieval reagent;
contacting the sample with a probe of the system according to any of claims 1 - 3 , 4 , and 5 - 8 , under conditions sufficient that the probe hybridizes to the target RNA in the sample;
rinsing the sample to remove unbound probe; and
detecting the target RNA by making visible the detectable moiety.

10. The method of claim 9 , wherein the method uses conditions that preserve cell morphology.

11. 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, the 2′-O-methyl RNA probe is conjugated with at least one detectable moiety disposed at either the 5′ end or the 3′ end, wherein the detectable moiety comprises a hapten, and is between 15 to 30 nucleotides in length, wherein the 2′-O-methyl RNA probe does not include any locked nucleic acid (LNA) substitutions, and wherein the target RNA is a microRNA (miRNA) target;
contacting the sample with a first anti hapten antibody conjugated with a first enzyme, wherein the first anti hapten antibody is specific for the 2′-O-methyl RNA probe;
contacting the sample with a reactive chromogen conjugate, wherein the reactive chromogen conjugate comprises a tyramide-hapten conjugate, wherein the first enzyme of the first anti hapten antibody binds the reactive chromogen conjugate to the first anti hapten 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, wherein the second enzyme catalyzes visibility of the chromogen, wherein the visibility of the chromogen is indicative of the target RNA.

12. The method of claim 11 , wherein the 2′-O-methyl RNA probe is conjugated with two detectable moieties.

13. The method of claim 11 , wherein the detectable moiety comprises dinitrophenol (DNP).

14. The method of claim 12 , wherein a first hapten is located at a 3′ end of the probe, and a second hapten is located at a 5′ end of the probe.

15. The method of claim 11 , wherein the reactive chromogen conjugate comprises a tyramide chromogen conjugate.

Assignees

• Ventana Med Syst Inc

Inventors

• Farrell Michael
• Day William
• Jiang Zeyu
• Pedata Anne

Classifications

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/178

  miRNA, siRNA or ncRNA · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q1/682

  Signal amplification · CPC title