Detection of target nucleic acids in a cellular sample

• US 9,783,841 B2
• Filed: 10/03/2013
• Issued: 10/10/2017
• Est. Priority Date: 10/04/2012
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Nolan, Garry P., Goltsev, Yury, Nguyen, Quan, Ma, Yunqing, Lai, Chunfai
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/045,698
• Publication Number:
  US 20140099637A1
• Time in Patent Office: 1,468 Days
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2527/149 Concentration of an enzyme

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.

Claims

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.

42. The method according to claim 1, wherein the permeabilization reagent comprises methanol.

47. The method according to claim 1, 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.

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.

43. The method according to claim 25, wherein the permeabilization reagent comprises methanol.

48. The method according to claim 25, wherein the aqueous post-fixation reagent comprises an aldehyde fixative.

52. The method according to claim 48, wherein the aqueous post-fixation reagent comprises at least one of:

formaldehyde and glutaraldehyde.

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.

44. The method according to claim 32, wherein the permeabilization reagent comprises methanol.

46. The method according to claim 32, wherein the aqueous post-fixation reagent comprises a cross-linking fixative.

49. The method according to claim 32, wherein the aqueous post-fixation reagent comprises an aldehyde fixative.

53. The method according to claim 49, wherein the aqueous post-fixation reagent comprises at least one of:

formaldehyde and glutaraldehyde.

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.

50. The method according to claim 45, wherein the aqueous post-fixation reagent comprises an aldehyde fixative.

54. The method according to claim 50, wherein the aqueous post-fixation reagent comprises at least one of:

formaldehyde and glutaraldehyde.