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 post-fixation reagent comprising at least one fixative selected from the group consisting of: a cross-linking fixative, an aldehyde, formaldehyde, glutaraldehyde, an imidoester, and an N-Hydroxysuccinimide (NHS) ester, (iv) a nuclease inhibitor, and (v) a nucleic acid detection agent comprising a signal producing system, 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 2 , wherein the nuclease inhibitor is an RNase inhibitor.
5. The method according to claim 1 , wherein the method further comprises contacting the cellular sample with a protein detection reagent.
6. The method according to claim 5 , wherein the protein detection reagent comprises a labeled binding member that specifically binds to a target protein.
7. The method according to claim 6 , wherein the labeled binding member comprises an antibody or binding fragment thereof.
8. The method according to claim 6 , wherein the label is a metal.
9. The method according to claim 6 , wherein the measuring comprises measuring the amount of label using mass cytometry.
10. The method according to claim 1 , wherein the signal producing system comprises a nucleic acid probe comprising a label that produces the signal.
11. The method according to claim 10 , wherein the label is a metal.
12. The method according to claim 11 , wherein the measuring comprises mass cytometry.
13. The method according to claim 1 , wherein the signal producing system comprises a signal amplification component.
14. The method according to claim 13 , wherein the signal producing system comprises a branched nucleic acid.
15. The method according to claim 1 , wherein the measuring comprises flow cytometry.
16. The method according to claim 1 , wherein the method comprises, after contacting the cellular sample with the nuclease inhibitor, storing the cellular sample for a period of time prior to the measuring step.
17. The method according to claim 16 , wherein the storing is in an aqueous buffer comprising a nuclease inhibitor.
18. The method according to claim 17 , wherein the storing is at a temperature below 0° C.
19. The method according to claim 1 , comprising contacting the cellular sample with a stimulating agent prior to contact with the fixation reagent.
20. The method according to claim 1 , wherein the method further comprises obtaining the cellular sample from a living animal.
21. The method according to claim 20 , wherein the living animal is a human.
22. The method according to claim 21 , wherein the method further comprises employing a result of the evaluating step to diagnose a state of the human.
23. The method according to claim 22 , wherein the state is a disease state.
24. The method according to claim 23 , wherein the disease state is a cellular proliferative disease state.
25. A method of obtaining a cellular sample, the method comprising:
obtaining a cellular sample comprising one or more cells in suspension from a living animal; and
contacting the cellular sample with, in order: (i) a fixation reagent, (ii) a permeabilization reagent, (iii) an aqueous post-fixation reagent comprising at least one fixative selected from the group consisting of: a cross-linking fixative, an aldehyde, formaldehyde, glutaraldehyde, an imidoester, and an N-Hydroxysuccinimide (NHS) ester, and (iv) a nuclease inhibitor
to produce a nuclease inhibitor-contacted hydrated/fixed suspended cellular sample.
26. The method according to claim 25 , wherein the nuclease inhibitor is an RNAse inhibitor.
27. The method according to claim 25 , 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 25 , wherein the method further comprises contacting the cellular sample with a stimulating agent.
31. The method according to claim 25 , wherein the living animal is a human.
32. 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 post-fixation reagent comprising at least one fixative selected from the group consisting of: a cross-linking fixative, an aldehyde, formaldehyde, glutaraldehyde, an imidoester, and an N-Hydroxysuccinimide (NHS) ester, and (iv) a nucleic acid detection agent comprising a signal producing system 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.
33. The method according to claim 32 , wherein the target nucleic acid is a ribonucleic acid (RNA).
34. The method according to claim 33 , wherein the RNA is a microRNA, a fusion gene transcript, or a splice variant.
35. The method according to claim 32 , wherein the nucleic acid detection agent comprises a signal producing system comprising a labeled nucleic acid probe.
36. The method according to claim 32 , wherein the signal producing system comprises a signal amplification component.
37. The method according to claim 36 , wherein the signal amplification component is a branched nucleic acid.
38. The method according to claim 32 , wherein the method further comprises contacting the cellular sample with a protein detection reagent.
39. The method according to claim 38 , wherein the protein detection reagent comprises a labeled binding member that specifically binds to a target protein.
40. The method according to claim 39 , wherein the labeled binding member comprises an antibody or binding fragment thereof.
41. The method according to claim 32 , wherein the measuring comprises flow cytometry.
42. The method according to claim 1 , wherein the permeabilization reagent comprises methanol.
43. The method according to claim 25 , wherein the permeabilization reagent comprises methanol.
44. The method according to claim 32 , wherein the permeabilization reagent comprises methanol.
45. A method of assaying a cellular sample that comprises one or more cells in suspension for the presence of a target RNA, the method comprising:
contacting the cellular sample with, in order: (i) an aqueous fixation reagent, (ii) a permeabilization reagent comprising methanol or ethanol, (iii) an aqueous post-fixation reagent comprising at least one cross-linking fixative, (iv) a nuclease inhibitor, and (v) 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.
46. The method according to claim 32 , wherein the aqueous post-fixation reagent comprises a cross-linking fixative.
47. The method according to claim 1 , wherein the aqueous post-fixation reagent comprises an aldehyde fixative.
48. The method according to claim 25 , wherein the aqueous post-fixation reagent comprises an aldehyde fixative.
49. The method according to claim 32 , wherein the aqueous post-fixation reagent comprises an aldehyde fixative.
50. The method according to claim 45 , wherein the aqueous post-fixation reagent comprises an aldehyde fixative.
51. The method according to claim 47 , wherein the aqueous post-fixation reagent comprises at least one of: formaldehyde and glutaraldehyde.
52. The method according to claim 48 , wherein the aqueous post-fixation reagent comprises at least one of: formaldehyde and glutaraldehyde.
53. The method according to claim 49 , wherein the aqueous post-fixation reagent comprises at least one of: formaldehyde and glutaraldehyde.
54. The method according to claim 50 , wherein the aqueous post-fixation reagent comprises at least one of: formaldehyde and glutaraldehyde.