Method for Detecting ncRna

• US 20080166707A1
• Filed: 04/20/2005
• Published: 07/10/2008
• Est. Priority Date: 04/20/2004
• Status: Active Grant

• Current Assignee: Jian Han
• Original Assignee: Jian Han
• Inventors: Han, Jian
• Granted Patent: US 7,811,759 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Described is a novel method for the detection of ncRNA molecules. The disclosed method is especially useful for the detection miRNA and siRNA. The method can be used to generate a profile of the ncRNA molecules present in a sample. In addition, using the methods of the present disclosure a ncRNA signature for a given disease or condition can be created. The ncRNA signature can be used for diagnostic purposes, therapeutic purposes and drug discovery purposes, as well as other uses.

Claims

1. A method for the simultaneous detection of a plurality of distinct target ncRNAs, said method comprising the steps of:

a. providing a RNA sample from a subject, said sample containing said plurality of target ncRNAs;

b. contacting said sample with a first oligonucleotide specific for each of said target ncRNAs to be detected under conditions appropriate to form a complex between said first oligonucleotides and said target ncRNAs, each of said first oligonucleotides comprising a first signal generator to generate a first detectable signal and each of said first oligonucleotides having a first Tm for binding each of said target ncRNAs that is substantially the same;

c. contacting said sample with a second oligonucleotide to bind for each of said target RNA to be detected under conditions appropriate to form a complex between said second oligonucleotides and said target ncRNAs, said second oligonucleotide comprising a second signal generator to generate a second detectable signal and each of said second oligonucleotides having a second Tm for binding each of said target ncRNAs that is substantially the same; and

d. determining the presence of said plurality of target ncRNA in said sample by measuring the first and second detectable signals.

2. The method of claim 1 where said ncRNA is a miRNA.

3. The method of claim 1 where said first detectable signal is different for each of said first oligonucleotides that binds a distinct ncRNA in said plurality of target ncRNAs.

4. The method of claim 1 where said first oligonucleotide and said second oligonucleotide are added simultaneously.

5. The method of claim 1 where said first and second oligonucleotides are added sequentially.

6. The method of claim 1 where each of said first Tms and each of said second Tms are substantially the same.

7. The method of claim 1 where at least one of said first oligonucleotide and said second oligonucleotide comprises at least one modified nucleotide.

8. The method of claim 7 where said modified nucleotide is a locked nucleic acid.

9. The method of claim 8 where each of said first Tms and each of said second Tms are substantially the same.

10. The method of claim 1 where said first and second detectable signals comprise an optical signature.

11. The method of claim 1 where said first and second detectable signals are a fluorescent signal.

12. The method of claim 1 where said first signal generator is a microsphere, said microsphere generating said first detectable signal.

13. The method of claim 12 where said first detectable signal is an optical signature.

14. The method of claim 12 where said first detectable signal is a fluorescent signal.

15. The method of claim 1 where said first oligonucleotide is 8-12 nucleotides in length and said second oligonucleotide is 8-12 nucleotides in length.

16. The method of claim 1 where said method is used to generate a profile of said target ncRNAs present in said sample.

17. The method of claim 1 where said sample is derived from a human subject.

18. The method of claim 1 where said RNA sample is not size fractionated to increase the proportion of a lower molecular weight RNA species.

34. The method of claim 1 where said method is used to generate a profile of said target ncRNAs present in said sample.

19. A method for determining a profile of a plurality of target ncRNA molecules in a RNA sample, said method comprising the steps of:

a. providing said RNA sample from a subject, said sample containing said plurality of target ncRNAs;

b. contacting said sample with a first oligonucleotide specific for each of said target ncRNAs to be detected under conditions appropriate to form a complex between said first oligonucleotides and said target ncRNAs, each of said first oligonucleotides comprising a first signal generator to generate a first detectable signal and each of said first oligonucleotides having a first Tm for binding each of said target ncRNAs that is substantially the same;

c. contacting said sample with a second oligonucleotide to bind for each of said target RNA to be detected under conditions appropriate to form a complex between said second oligonucleotides and said target ncRNAs, said second oligonucleotide comprising a second signal generator to generate a second detectable signal and each of said second oligonucleotides having a second Tm for binding each of said target ncRNAs that is substantially the same; and

d. determining the presence of said plurality of target ncRNA in said sample by measuring the first and second detectable signals.

20. The method of claim 19 where said ncRNA is a miRNA.

21. The method of claim 19 where said first detectable signal is different for each of said first oligonucleotides that binds a distinct ncRNA in said plurality of target ncRNAs.

22. The method of claim 19 where said first oligonucleotide and said second oligonucleotide are added simultaneously.

23. The method of claim 19 where said first and second oligonucleotides are added sequentially.

24. The method of claim 19 where each of said first Tms and each of said second Tms are substantially the same.

25. The method of claim 19 where at least one of said first oligonucleotide and said second oligonucleotide comprises at least one modified nucleotide.

26. The method of claim 25 where said modified nucleotide is a locked nucleic acid.

27. The method of claim 26 where each of said first Tms and each of said second Tms are substantially the same.

28. The method of claim 19 where said first and second detectable signals comprise an optical signature.

29. The method of claim 19 where said first and second detectable signals are a fluorescent signal.

30. The method of claim 19 where said first signal generator is a microsphere, said microsphere generating said first detectable signal.

31. The method of claim 30 where said first detectable signal is an optical signature.

32. The method of claim 30 where said first detectable signal is a fluorescent signal.

33. The method of claim 19 where said first oligonucleotide is 8-12 nucleotides in length and said second oligonucleotide is 8-12 nucleotides in length.

35. The method of claim 19 where said sample is derived from a human subject.

36. The method of claim 19 where said RNA sample is not size fractionated to increase the proportion of a lower molecular weight RNA species.

37. A method of creating a signature ncRNA profile for a disease or condition, said method comprising the steps of:

a. obtaining a ncRNA profile from a sample characterized as having said disease or condition to create a first profile, said profile being obtained by the methods of claim 19;

b. obtaining a ncRNA profile from a sample characterized as normal to create a second profile said profile being obtained by the methods of claim 19; and

c. identifying one or more ncRNA molecules in said first profile that distinguish said first profile from said second profile.

38. The method of claim 37 where said ncRNA is a miRNA.

39. The method of claim 37 where said disease is glioma or breast cancer.

40. The method of claim 37 where said subject is a human.

41. A method of diagnosing a subject with a disease or condition, said method comprising the steps of:

a. determining a subject ncRNA profile from said subject;

b. comparing said subject ncRNA profile to a ncRNA profile for said disease or condition, said subject ncRNA profile and said ncRNA profile for said disease or condition being determined according to the method of claim 19;

c. diagnosing said subject with said disease or condition based on a comparison of said subject ncRNA profile to said ncRNA profile for said disease or condition.

42. The method of claim 41 where said ncRNA is a miRNA.

43. The method of claim 41 where said disease is glioma or breast cancer.

44. The method of claim 41 where said subject is a human.

45. A method of screening to identify a gene involved in a disease or condition, said method comprising the steps of:

a. obtaining a signature ncRNA profile for said disease or condition, said signature ncRNA profile being obtained by the method of claim 37; and

b. determining a molecular target with which one said ncRNAs identified in signature ncRNA profile interact.

46. The method of claim 45 where said ncRNA is a miRNA.

47. The method of claim 45 where said disease is glioma or breast cancer.

48. The method of claim 45 where said subject is a human.

49. The method of claim 45 where said determining step is accomplished with the aid of an algorithm.

76. A method for creating a signature ncRNA profile for a disease state or condition, said method comprising:

a. determining a first ncRNA profile from a first source, said first source being characterized as being free from said disease state or condition;

b. determining a second ncRNA profile from a second source, said second source characterized as being positive for said disease state or condition, said first and second ncRNA profile being obtained according to the method of claim 19;

c. comparing said first and second ncRNA profiles and identifying those ncRNA molecules that are altered in said second ncRNA profile to create a signature ncRNA profile for said disease state or condition.

77. The method of claim 76 where said disease state is breast cancer and said signature ncRNA profile comprises mir-107, mir-15B and mir-103.

78. The method of claim 77 where said signature ncRNA further comprises at least one of:

mir-17-5p, mir-16, mir-126, mir-141, mir-193, mir29a, mir-195, mir-26a, mir20, mir128b, mir217 and mir219.

79. The method of claim 76 where said disease state is glioma and said signature miRNA profile comprises mir-141, mir-23b and mir-17-3p.

80. The method of claim 79 where said signature ncRNA further comprises at least one of:

mir-143, mir-15b, mir-293 and mir-320.

81. The signature miRNA profile of claim 76 where said profile is used to diagnose a subject in need of said diagnosis.

82. The signature miRNA profile of claim 76 where said profile is used to identify candidate therapeutic targets for drug development.

50. (canceled)

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57. (canceled)

58. A method for the detection of a target ncRNA, said method comprising the steps of:

a. providing a RNA sample from a subject, said sample containing said target ncRNA

b. contacting said sample with a first oligonucleotide specific for said target RNA under conditions appropriate to form a complex between said first oligonucleotide and said target ncRNA, said first oligonucleotide comprising a first signal generator to generate a first detectable signal;

c. contacting said sample with a second oligonucleotide specific for said target RNA under conditions appropriate to form a complex between said second oligonucleotide and said target ncRNA, said second oligonucleotide comprising a second signal generator to generate a second detectable signal; and

d. determining the presence of said target ncRNA in said sample by measuring the first and second detectable signals.

59. The method of claim 58 where said first oligonucleotide binds a first complementary sequence on said ncRNA and has a first Tm for binding to said first complementary sequence and said second oligonucleotide binds a second complementary sequence on said ncRNA and has a second Tm for binding to a second complementary sequence, said first Tm and said second Tm being substantially the same.

60. The method of claim 58 where said first oligonucleotide and said second oligonucleotide are added simultaneously.

61. The method of claim 58 where said first and second oligonucleotides are added sequentially.

62. The method of claim 58 where at least one of said first oligonucleotide and said second oligonucleotide comprises at least one modified nucleotide.

63. The method of claim 62 where said modified nucleotide is a locked nucleic acid.

64. The method of claim 58 where said first oligonucleotide binds a first complementary sequence on said ncRNA and has a first Tm for binding to said first complementary sequence and said second oligonucleotide binds a second complementary sequence on said ncRNA and has a second Tm for binding to a second complementary sequence, said first Tm and said second Tm being substantially the same.

65. The method of claim 58 where said first and second detectable signals comprise an optical signature.

66. The method of claim 58 where said first and second detectable signals are a fluorescent signal.

67. The method of claim 58 where said first signal generator is a microsphere, said microsphere generating said first detectable signal.

68. The method of claim 67 where said first detectable signal is an optical signature.

69. The method of claim 68 where said first detectable signal is a fluorescent signal.

70. The method of claim 58 where said first oligonucleotide is 8-12 nucleotides in length and said second oligonucleotide is 8-12 nucleotides in length.

71. The method of claim 58 where said method is used to generate a profile of said ncRNA molecules present in said sample.

72. The method of claim 58 where said sample is derived from a human subject.

73. The method of claim 58 where said method is used in a multiplex detection reaction.

74. The method of claim 58 where said RNA sample is not size fractionated to increase the proportion of a lower molecular weight RNA species.

75. The method of claim 58 where said ncRNA is a miRNA.