Diagnostic histopathology using multiplex gene expression FISH
View Patent ↗The present invention relates to a method for detecting one or more nascent RNAs in a tissue sample using FISH. In the method, a plurality of probes (8 to 82) may be used to detect a single species of nascent RNA. Further, a plurality of nascent RNA species may be detected simultaneously using between 8 and 82 probes for each nascent RNA. The invention comprises, in addition, methods of preparing a sample for nascent RNA detection by reducing autofluorescence of the tissue sample. These techniques may be synergistically combined to achieve significantly improved results.
1. A method for the in situ detection of one or more nascent RNA species in a human paraffin embedded tissue sample comprising the steps of:
a) performing autofluorescence reduction on the human paraffin embedded tissue sample by pressure cooking the tissue sample at a temperature of about 125° C., at a pressure of between 20 to 24 PSI and for a period of 10 minutes to 1 hour to produce a pressure cooked sample, and then treating said pressure cooked sample with ammonia-ethanol at a concentration of 0.1% to 0.5% for between 10 and 50 minutes and sodium borohydride at a concentration of 1% to 5% for between 10 and 50 minutes in any order;
b) hybridizing at least one labeled nucleic acid probe specific for one or more nascent RNA species to the tissue sample;
c) detecting the at least one labeled nucleic acid probe in the tissue sample thereby detecting the presence of the one or more nascent RNA species.
2. The method of claim 1 wherein the tissue sample is from a neoplastic or preneoplastic tissue.
3. The method of claim 1 wherein the ammonia-ethanol is in a concentration of 0.25%.
4. The method of claim 1 wherein treating the pressure cooked sample with ammonia-ethanol comprises contacting the pressure cooked sample with ammonia-ethanol for 10 to 30 minutes.
5. The method of claim 1 wherein treating the pressure cooked sample with ammonia-ethanol comprises contacting the pressure cooked sample with ammonia-ethanol for 40 minutes.
6. The method of claim 1 wherein treating the sample with sodium borohydride comprises contacting the pressure cooked sample with sodium borohydride for 20 minutes.
7. The method of claim 1 wherein the at least one labeled nucleic acid probe is attached to at least one fluorescent moiety.
8. The method of claim 7 wherein the fluorescent moiety is selected from the group consisting of FITC, Cy3, Cy3.5, Cy5, Cy5.5 and DAPI.
9. The method of claim 7 wherein each of said nascent RNA species hybridizes to a plurality of probes with a unique combination of fluorescent moieties for each of said nascent RNA species.
10. The method of claim 7 wherein each of said nascent RNA species hybridizes to 8 or more probes.
11. The method of claim 7 wherein each of said nascent RNA species hybridizes to 12 or more probes.
12. The method of claim 1 wherein the labeled nucleic acid probe comprises a nucleotide sequence of an intron, a 5′ untranslated region or a 3′ untranslated region of a nascent RNA.
13. The method of claim 1 wherein the labeled nucleic acid probe is specific for a 5′ region of the nascent RNA species.
14. The method of claim 1 wherein the labeled nucleic acid probe is specific for a 3′ region of the nascent RNA species.
15. The method of claim 1 further comprising a step of staining the sample after the detecting step.
16. The method of claim 1 wherein the detecting step comprises the step of detecting the labeled nucleic acid probe in the nucleus of a cell in the tissue sample.
17. The method of claim 1 wherein the detecting step detects the labeled nucleic acid probe within 50 micron of its location.
18. The method of claim 1 further comprising the step of determining the location of a chromosome in said tissue sample.
19. The method of claim 1 which is performed in the absence of any added protease.
20. The method of claim 18 wherein determining the location of a chromosome comprises the steps of
(1) hybridizing at least one labeled chromosome specific probe to said tissue sample;
(2) detecting said at least one labeled chromosome specific probe in at least one cell in said tissue sample.
21. The method of claim 20 wherein the step of detecting the labeled chromosome specific probe detects the labeled chromosome specific probe within 50 micron of its location.
22. The method of claim 18 wherein said step of determining the location of a chromosome is performed after step a), after step b), or after step c).