Detection of target nucleic acids in a cellular sample

Patent metadata

Field	Value
Publication number	US-9783841-B2
Application number	US-201314045698-A
Country	US
Kind code	B2
Filing date	Oct 3, 2013
Priority date	Oct 4, 2012
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

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.

First claim

What is claimed is:

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 , wherei

Assignees

• Univ Leland Stanford Junior

Inventors

• Nolan Garry P
• Goltsev Yury
• Nguyen Quan
• Ma Yunqing
• Lai Chunfai

Classifications

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q1/6813 Primary

  Hybridisation assays · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title

• C12Q2527/149

  Concentration of an enzyme · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title