Detection of target nucleic acids in a cellular sample
Methods of assaying cells of a cellular sample for the presence of a target nucleic acid are provided. Aspects of the methods include evaluating a cellular sample that has been contacted with a nuclease inhibitor for the presence of a target nucleic acid. Also provided are devices and kits that find use in practicing the methods described herein.
1. A method of assaying a cellular sample that comprises one or more cells in suspension for the presence of a target nucleic acid, the method comprising:
contacting the cellular sample with, in order: (i) a fixation reagent; (ii) a permeabilization reagent; (iii) an aqueous fixative comprising one or more fixation reagents selected from the group consisting of: a cross-linking fixative, a precipitating fixative, an oxidizing fixative, and a mercurial fixative; and (iv) a nucleic acid detection agent comprising a signal producing system that comprises a branched nucleic acid signal amplification component, to produce a suspended hydrated/fixed detection agent-contacted cellular sample; and
measuring a signal produced by the signal producing system to evaluate the suspended hydrated/fixed detection agent-contacted cellular sample for the presence of the target nucleic acid.
2. The method according to claim 1 , wherein the target nucleic acid is a ribonucleic acid (RNA).
3. The method according to claim 2 , wherein the RNA is a microRNA, a fusion gene transcript, or a splice variant.
4. The method according to claim 1 , wherein the method further comprises contacting the cellular sample with a protein detection reagent.
5. The method according to claim 4 , wherein the protein detection reagent comprises a labeled binding member that specifically binds to a target protein.
6. The method according to claim 5 , wherein the labeled binding member comprises an antibody or binding fragment thereof.
7. The method according to claim 6 , wherein the label is a metal.
8. The method according to claim 6 , wherein the measuring comprises measuring the amount of label using mass cytometry.
9. The method according to claim 1 , wherein the signal producing system comprises a nucleic acid probe comprising a label that produces the signal.
10. The method according to claim 9 , wherein the label is a metal.
11. The method according to claim 10 , wherein the measuring comprises mass cytometry.
12. The method according to claim 1 , wherein the measuring comprises flow cytometry.
13. The method according to claim 1 , wherein the method comprises, after contacting the cellular sample with the aqueous fixative, storing the cellular sample for a period of time prior to the measuring step.
14. The method according to claim 13 , wherein the storing is in an aqueous buffer comprising a nuclease inhibitor.
15. The method according to claim 14 , wherein the storing is at a temperature below 0° C.
16. The method according to claim 1 , comprising contacting the cellular sample with a stimulating agent prior to contact with the fixation reagent.
17. The method according to claim 1 , wherein the method further comprises obtaining the cellular sample.
18. The method according to claim 1 , wherein the cellular sample is a human cellular sample.
19. The method according to claim 18 , wherein the method further comprises employing a result of the evaluating step to diagnose a state of the human.
20. The method according to claim 18 , wherein the state is a disease state.
21. The method according to claim 20 , wherein the disease state is a cellular proliferative disease state.
22. The method according to claim 1 , further comprising contacting the cellular sample with a nuclease inhibitor after step (iii).
23. The method according to claim 1 , wherein the permeabilization reagent comprises methanol.
24. The method according to claim 1 , wherein the aqueous fixative comprises an aldehyde fixative.
25. The method according to claim 1 , wherein the aqueous fixative comprises at least one of: formaldehyde and glutaraldehyde.
26. A method comprising:
contacting a cellular sample that comprises one or more cells in suspension with, in order: (i) a fixation reagent; (ii) a permeabilization reagent; (iii) an aqueous fixative comprising one or more fixation reagents selected from the group consisting of: a cross-linking fixative, a precipitating fixative, an oxidizing fixative, and a mercurial fixative;
to produce a hydrated/fixed suspended cellular sample.
27. The method according to claim 26 , wherein the method further comprises contacting the cellular sample with a protein detection reagent.
28. The method according to claim 27 , wherein the protein detection reagent comprises a labeled binding member that specifically binds to a target protein.
29. The method according to claim 28 , wherein the labeled binding member comprises an antibody or binding fragment thereof.
30. The method according to claim 26 , wherein the method further comprises contacting the cellular sample with a stimulating agent.
31. The method according to claim 26 , wherein the cellular sample is a human cellular sample.
32. The method according to claim 26 , further comprising contacting the cellular sample with a nuclease inhibitor after step (iii).
33. The method according to claim 26 , wherein the permeabilization reagent comprises methanol.
34. The method according to claim 26 , wherein the aqueous fixative comprises an aldehyde fixative.
35. The method according to claim 26 , wherein the aqueous fixative comprises at least one of: formaldehyde and glutaraldehyde.
36. The method according to claim 26 , wherein the permeabilization reagent comprises methanol.
37. A method of assaying a cellular sample that comprises one or more cells in suspension for the presence of a target nucleic acid, the method comprising:
contacting the cellular sample with, in order: (i) a first aqueous fixative; (ii) a permeabilization reagent; (iii) a second aqueous fixative comprising one or more fixation reagents selected from the group consisting of: a cross-linking fixative, a precipitating fixative, an oxidizing fixative, and a mercurial fixative; and (iv) a nucleic acid detection agent comprising a signal producing system that comprises a branched nucleic acid, to produce a suspended hydrated/fixed detection agent-contacted cellular sample; and
measuring a signal produced by the signal producing system to evaluate the suspended hydrated/fixed detection agent-contacted cellular sample for the presence of the target RNA, wherein the measuring comprises flow cytometry.
38. The method according to claim 37 , wherein the first and/or second aqueous fixative comprises an aldehyde fixative.
39. The method according to claim 37 , wherein the first and/or second aqueous fixative comprises at least one of: formaldehyde and glutaraldehyde.
40. The method according to claim 37 , further comprising contacting the cellular sample with a nuclease inhibitor after step (iii).
41. The method according to claim 37 , wherein the permeabilization reagent comprises methanol.
42. The method according to claim 1 , wherein the cross-linking fixative is an imidoester or a N-Hydroxysuccinimide (NHS) ester.
43. The method according to claim 26 , w wherein the cross-linking fixative is an imidoester or a N-Hydroxysuccinimide (NHS) ester.
44. The method according to claim 37 , wherein the cross-linking fixative is an imidoester or a N-Hydroxysuccinimide (NHS) ester.
45. The method according to claim 1 , wherein the precipitating fixative is an alcohol, acetic acid, or acetone.
46. The method according to claim 26 , wherein the precipitating fixative is an alcohol, acetic acid, or acetone.
47. The method according to claim 37 , wherein the precipitating fixative is an alcohol, acetic acid, or acetone.