Detection of target nucleic acids in a cellular sample

• US 10,689,687 B2
• Filed: 08/30/2017
• Issued: 06/23/2020
• Est. Priority Date: 10/04/2012
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Nolan, Garry P., Goltsev, Yury, Nguyen, Quan, Ma, Yunqing, Lai, Chunfai
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/691,580
• Publication Number:
  US 20170362642A1
• Time in Patent Office: 1,028 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 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.

42. The method according to claim 1, 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.

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.

43. The method according to claim 26, w wherein the cross-linking fixative is an imidoester or a N-Hydroxysuccinimide (NHS) ester.

46. The method according to claim 26, wherein the precipitating fixative is an alcohol, acetic acid, or acetone.

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.

44. The method according to claim 37, wherein the cross-linking fixative is an imidoester or a N-Hydroxysuccinimide (NHS) ester.

47. The method according to claim 37, wherein the precipitating fixative is an alcohol, acetic acid, or acetone.