Automated RNA detection using labeled 2′-O-methyl RNA oligonucleotide probes and signal amplification systems
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.
1. A system for bright field in situ hybridization comprising:
a probe set comprising at least two first target probes, wherein each of the at least two first target probes are specific to different portions of a first target RNA, wherein each of the at least two first target probes are conjugated to least one detectable moiety, and wherein the at least two first target probes consist of 2′-O-methyl nucleotides; and
a reactive chromogen conjugate system effective for signal amplification, wherein each reactive chromogen conjugate system is adapted to bind to the at least one detectable moiety of each first target probe of the at least two first target probes.
2. The system of claim 1 , wherein the at least two first target probes each comprise between 15 to 30 nucleotides, between 20 to 50 nucleotides, between 40 to 80 nucleotides, between 20 to 100 nucleotides, or between 20 to 200 nucleotides in length.
3. The system of claim 1 , wherein the at least two first target probes are each conjugated with at least two detectable moieties, at least three detectable moieties, at least four detectable moieties, or at least five detectable moieties.
4. The system of claim 1 , wherein the detectable moiety comprises a hapten.
5. The system of claim 4 , wherein the hapten is selected from the group consisting of dinitrophenol (DNP), nitropyrazole, hydroxyquinoxaline, thiazolesulfonamide, nitrocinnamic acid, rotenone, 7-(diethylamino) coumarin-3-carboxylic acid, benzodiazepine, and benzofuran.
6. The system of claim 1 , wherein the reactive chromogen conjugate system comprises a tyramide-hapten conjugate.
7. The system of claim 1 , wherein the at least two first target probes each comprise at least one detectable moiety per 20 base pairs of the target probe.
8. The system of claim 1 , further comprising a means of making a target microRNA visible.
9. The system of claim 8 , wherein the means of making the target microRNA visible comprises means for contacting the probes with the reactive chromogen conjugate system specific to the detectable moieties of the probes, wherein the reactive chromogen conjugate system emits a color.
10. The system of claim 8 , further comprising a means of visualizing the target microRNA, wherein the detectable moieties are made visible by the reactive chromogen conjugate system, the visibility of the detectable moieties being indicative of the target microRNA.
11. The system of claim 10 , wherein the means of visualizing the target microRNA comprises a bright field microscope.
12. 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 and 4-11 under conditions sufficient that the target 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.
13. The method of claim 12 , wherein the method uses conditions that preserve cell morphology.
14. 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 at least two first target probes, wherein each of the at least two first target probes are specific to different portions of a first target RNA, wherein each of the at least two first target probes are conjugated to least one detectable moiety, and wherein the at least two first target probes consist of 2′-O-methyl nucleotides;
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 at least two target 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/or 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.
15. A method of in situ hybridization comprising:
contacting a sample with at least two first target probes, where each of the at least two first target probes consist of 2′-O-methyl nucleotides and wherein each of the at least two first target probes are specific to different portions of a first target RNA in the sample; wherein the sample is contacted with the at least two first target probes under conditions sufficient that the at least two first target probes hybridizes to the first target RNA in the sample, wherein the at least two first target probes are each conjugated with at least one detectable moiety disposed at either the 5′ end or the 3′ end of the at least two first target probes, and wherein each of the at least two first target probes are 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 at least one detectable moiety disposed at either the 5′ end or the 3′ end of the at least two first target probes;
contacting the sample with 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, wherein the second enzyme catalyzes visibility of the chromogen, wherein visibility of the chromogen is indicative of the target RNA.
16. The method of claim 15 , wherein each of the at least two target probes are conjugated with to two detectable moieties.
17. The method of claim 15 , wherein the detectable moiety comprises dinitrophenol (DNP).
18. The method of claim 16 , 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.
19. The method of claim 15 , wherein the reactive chromogen conjugate comprises a tyramide chromogen conjugate.
20. The system of claim 1 , further comprising at least one second target probe, wherein the at least one second target probe is specific to at least a portion of a second target RNA, wherein the at least one second target probe consists of 2′O-methyl RNA oligonucleotides; and wherein the system further comprises a second reactive chromogen conjugate system.
21. The system of claim 20 , wherein the second reactive chromogen conjugate system comprises a second tyramide-happen conjugate system.