RNA amplification method

• US 6,271,002 B1
• Filed: 10/04/1999
• Issued: 08/07/2001
• Est. Priority Date: 10/04/1999
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Rosetta Inpharmatics Incorporated
• Inventors: Schelter, Janell M., Linsley, Peter S.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US09/411,074
• Time in Patent Office: 673 Days
• Field of Search: 435/91.1, 435/91.2, 435/6, 435/5, 435/4, 435/7.1, 435/288, 536/24.3, 536/23.4, 436/501
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

The present invention relates to methods and kits for amplification of mRNA using a primer in PCR that contains an RNA polymerase promoter. The invention provides methods for amplification and detection of RNA derived from a population of cells, preferably eukaryotic cells and most preferably mammalian cells, which methods preserve fidelity with respect to sequence and transcript representation, and additionally enable amplification of extremely small amounts of mRNA, such as might be obtained from 10⁶ mammalian cells. In typical embodiments of the invention, an RNA polymerase promoter (RNAP) is incorporated into ds cDNA by priming cDNA amplification by polymerase chain reaction (PCR) with an RNAP-containing primer. Following less than 20 cycles of PCR, the resultant RNAP-containing ds cDNA is transcribed into RNA using an RNA polymerase capable of binding to the RNAP introduced during cDNA synthesis. This combination of PCR and in vitro transcription (IVT) enables the generation of a relatively large amount of RNA from a small starting number of cells without loss of fidelity. RNAs generated using this method may be labeled and employed to profile gene expression in different populations of cells, e.g., by use of a polynucleotide microarray.

Claims

1. A method for amplifying at least one mRNA in a sample containing a plurality of different mRNAs comprising:

(a) synthesizing first strand cDNA by contacting under conditions conducive to reverse transcriptase activity at least one mRNA in said sample with (i) reverse transcriptase, and (ii) a first primer that is sufficiently complementary to a sequence in the mRNA so as to prime synthesis in a direction toward the 5′

end of the mRNA;

(b) synthesizing double-stranded cDNA by contacting under conditions conducive to DNA polymerase activity the first strand cDNA with (i) a first DNA polymerase, and (ii) a second primer that is sufficiently complementary to a sequence 5′

to said first primer sequence in said first strand cDNA so as to prime synthesis in a direction toward said first primer sequence;

wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;

(c) amplifying the double-stranded cDNA by subjecting the double-stranded cDNA to a single round of polymerase chain reaction (hereinafter “

PCR”

) of 20 cycles or less, wherein DNA is synthesized by use of a second DNA polymerase and a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each being sufficiently complementary to a different strand of said double-stranded cDNA so as to prime synthesis in a template-dependent manner, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation; and

(d) transcribing resultant amplified DNA into cRNA by contacting the amplified DNA with an RNA polymerase specific for said RNA polymerase promoter sequence introduced in step (c) under conditions conducive to RNA polymerase activity, such that cRNA is produced.

2. The method of claim 1 wherein in step (c) or less cycles of PCR are carried out.

3. The method of claim 1 wherein said first primer is a mixture of primers that prime synthesis in a direction toward the 5′

end of substantially all the mRNAs in the sample and said second primer is a mixture of primers that prime synthesis of double-stranded cDNA from substantially all said first strand cDNAs produced in step (a).

4. The method of claim 2 wherein said first primer is a mixture of primers that prime synthesis in a direction toward the 5′

end of substantially all the mRNAs in the sample and said second primer is a mixture of primers that prime synthesis of double-stranded cDNA from substantially all said first strand cDNAs produced in step (a).

5. The method of claim 3 wherein said first primer is a mixture of first primers, each primer comprising an oligo (dT) sequence and a 3′

end sequence of 1-5 nucleotides, and said mixture of first primers comprising primers having an A, a G, or a C nucleotide present in each position of said 3′

end sequence.

6. The method of claim 4 wherein said first primer is a mixture of first primers, each primer comprising an oligo (dT) sequence and a 3′

end sequence of 1-5 nucleotides, and said mixture of first primers comprising primers having an A, a G, or a C nucleotide present in each position of said 3′

end sequence.

7. The method of claim 5 wherein each primer in said mixture of first primers further comprises at its 5′

end a selected sequence of 5-12 nucleotides.

8. The method of claim 6 wherein each primer in said mixture of first primers further comprises at its 5′

end a selected sequence of 5-12 nucleotides.

9. The method of claim 3 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence.

10. The method of claim 4 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence.

11. The method of claim 7 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence.

12. The method of claim 8 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C or a T nucleotide present in each position of said 3′

end sequence.

13. The method of claim 1 or 2 wherein said forward primer comprises said RNA polymerase promoter-sequence.

14. The method of claim 1 or 2 wherein said reverse primer comprises said RNA polymerase promoter sequence.

15. The method of claim 11 wherein said forward primer comprises a 3′

end sequence of 8-12 nucleotides that is the same as the 5′

end sequence of 8-12 nucleotides of the second primer, and said reverse primer comprises a 3′

end sequence of 5-12 nucleotides that is the same as the 5′

end sequence of 5-12 nucleotides of the first primer.

16. The method of claim 12 wherein said forward primer comprises a 3′

end sequence of 8-12 nucleotides that is the same as the 5′

end sequence of 8-12 nucleotides of the second primer, and said reverse primer comprises a 3′

end sequence of 5-12 nucleotides that is the same as the 5′

end sequence of 5-12 nucleotides of the first primer.

17. The method of claim 15 wherein said reverse primer comprises said RNA polymerase promoter sequence.

18. The method of claim 15 wherein said forward primer comprises said RNA polymerase promoter sequence.

19. The method of claim 1 wherein said sample contains total RNA or total mRNA from cells.

20. The method of claim 2, 3, 15 or 16 wherein said sample contains total RNA or total mRNA from cells.

21. The method of claim 19 wherein said cells are yeast cells.

22. The method of claim 19 wherein said cells are mammalian cells.

23. The method of claim 1 wherein said sample contains total RNA from 1×

10⁶ cells or less.

24. The method of claim 2 wherein said sample contains total RNA from 1×

10⁶ cells or less.

25. The method of claim 3, 15, 16, 17 or 18 wherein said sample contains total RNA from 1×

10⁶ cells or less.

26. The method of claim 1, wherein said first DNA polymerase is E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.

27. The method of claim 1, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence and the RNA polymerase is T7 RNA polymerase.

28. The method of claim 1, wherein the RNA polymerase promoter sequence is a T3 RNA polymerase promoter sequence and the RNA polymerase is T3 RNA polymerase.

29. The method of claim 1, wherein the RNA polymerase promoter sequence is an SP6 RNA polymerase promoter sequence and the RNA polymerase is SP6 RNA polymerase.

30. The method of claim 1, which further comprises labeling the transcribed cRNA with a label.

31. The method of claim 30, wherein the label is a fluorescent, radioactive, enzymatic, hapten, biotin, or digoxygenin label.

32. The method of claim 31, wherein the label is fluorescent.

33. The method of claim 32 wherein the fluorescent label is fluorescein isothiocyanate, lissamine, Cy3, Cy5, or rhodamine 110.

34. The method of claim 32, wherein a first aliquot of the cRNA is labeled with a first fluorophore having a first emission spectrum, and a second aliquot of the cRNA is labeled with a second fluorophore with a second emission spectrum differing from that of the first emission spectrum.

35. The method of claim 32, wherein the first fluorophore is Cy3 and the second fluorophore is Cy5.

36. The method of claim 1 or 2, further comprising, after the transcribing step, determining the presence or absence of a preselected target mRNA in said sample.

37. The method of claim 1, wherein the mRNA is derived from a population of cells.

38. The method of claim 37, wherein the population of cells is a eukaryotic population of cells.

39. The method of claim 38, wherein the eukaryotic population of cells is 106 or fewer mammalian cells.

40. The method of claim 1, wherein the first, second, forward, and reverse primers are each 24-48 nucleotides in length.

41. The method of claim 1, wherein the mRNA is extracted from at least one cell of interest, and further comprising contacting the cRNA produced in step (d) with an array containing one or more species of polynucleotide positioned at preselected sites on the array, under conditions conducive to hybridization;

and detecting any hybridization that occurs between said one or more species of polynucleotide and said cRNA.

42. The method of claim 2, wherein the mRNA is extracted from at least one cell of interest, and further comprising contacting the cRNA produced in step (d) with an array containing one or more species of polynucleotide positioned at preselected sites on the array, under conditions conducive to hybridization;

and detecting any hybridization that occurs between said one or more species of polynucleotide and said cRNA.

43. The method of claim 15, 16, 17 or 18, wherein the mRNA is extracted from at least one cell of interest, and further comprising contacting the cRNA produced in step (d) with an array containing one or more species of polynucleotide positioned at preselected sites on the array, under conditions conducive to hybridization;

and detecting any hybridization that occurs between said one or more species of polynucleotide and said cRNA.

44. The method of claim 1 wherein said sample contains at least 10,000 different mRNAs.

75. The method of claim 41 wherein the array comprises a support with at least one surface and at least 100 different polynucleotide probes, each different polynucleotide probe comprising a different nucleotide sequence and being attached to the surface of the support in a different, selected location on said surface.

77. The method of claim 41 wherein the nucleotide sequence of each of the different polynucleotide probes is in the range of 40-80 nucleotides in length.

78. The method of claim 41 wherein the polynucleotide probes comprise cDNA sequences.

79. The method of claim 41 wherein the array comprises polynucleotide probes of at least 2,000 different sequences.

80. The method of claim 41 wherein the nucleotide sequence of the polynucleotide probes are specific for at least 50% of the genes in the genome of the cells of interest.

81. The method of claim 41 wherein the polynucleotide probes hybridize to at least 10,000 different polynucleotide sequences.

82. The method of claim 41 wherein the array has at least 1,000 polynucleotide probes per 1 cm².

100. The method of claim 1 or 45 wherein said first primer comprises an oligo dT sequence.

101. The method of claim 1 or 45 wherein said first primer comprises a mixture of primers having an A, a G, a C, or a T nucleotide present in each position of the sequence of said primer.

102. The method of claim 1, 3, 4, 45, 48, or 49 wherein said second DNA polymerase of said amplifying step is thermostable.

45. A method for comparing the presence or amount of at least one mRNA of interest in a first sample and in a second sample, said first sample and said second sample each containing a plurality of different mRNAs from one or more cells, comprising:

(a) synthesizing first strand cDNA by contacting under conditions conducive to reverse transcriptase activity at least one mRNA in said first sample with (i) reverse transcriptase, and (ii) a first primer that is sufficiently complementary to a sequence in the mRNA so as to prime synthesis in a direction toward the 5′

end of the mRNA;

(b) synthesizing double-stranded cDNA by contacting under conditions conducive to DNA polymerase activity the first strand cDNA with (i) a first DNA polymerase, and (ii) a second primer that is sufficiently complementary to a sequence 5′

to said first primer sequence in said first strand cDNA so as to prime synthesis in a direction toward said first primer sequence;

wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;

(c) amplifying the double-stranded cDNA by subjecting the double-stranded cDNA to a single round of PCR of 20 cycles or less, wherein DNA is synthesized by use of a second DNA polymerase and a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each being sufficiently complementary to a different strand of said double-stranded cDNA so as to prime synthesis in a templatedependent manner, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;

(d) transcribing resultant amplified DNA into cRNA by contacting the amplified DNA with an RNA polymerase specific for said RNA polymerase promoter sequence introduced in step (c) under conditions conducive to RNA polymerase activity, such that cRNA is produced;

(e) labeling the cRNA produced in step (d) with a first label;

(f) repeating steps (a)-(d) with said second sample;

(g) labeling the cRNA produced in step (f) with a second label distinguishable from said first label;

(h) detecting or measuring the mRNA of interest in the first sample by contacting the cRNA labeled with said first label with a polynucleotide probe capable of hybridizing to said cRNA of the mRNA of interest under conditions conducive to hybridization; and

detecting any hybridization that occurs between said probe and said cRNA;

(i) detecting or measuring the mRNA of interest in the second sample by contacting the cRNA labeled with said second label with said polynucleotide probe capable of hybridizing to said cRNA of the mRNA of interest under conditions conducive to hybridization; and

detecting any hybridization that occurs between said probe and said cRNA; and

(j) comparing the mRNA of interest detected or measured in said first sample with the mRNA of interest detected or measured in said second sample.

46. The method of claim 45 wherein in said amplifying steps 10 or less cycles of PCR are carried out.

47. The method of claim 45 wherein said first primer is a mixture of primers that prime synthesis toward the 5′

end of substantially all the mRNAs in the sample and said second primer is a mixture of primers that prime synthesis of double-stranded cDNA from substantially all said first strand cDNAs produced in said steps of synthesizing first strand cDNA.

48. The method of claim 46 wherein said first primer is a mixture of primers that prime synthesis toward the 5′

end of substantially all the mRNAs in the sample and said second primer is a mixture of primers that prime synthesis of double-stranded cDNA from substantially all said first strand cDNAs produced in said steps of synthesizing first strand cDNA.

49. The method of claim 47 wherein said first primer is a mixture of first primers, each primer comprising an oligo (dT) sequence and a 3′

end sequence of 1-5 nucleotides, and said mixture of first primers comprising primers having an A, a G, or a C nucleotide present in each position of said 3′

end sequence.

50. The method of claim 48 wherein said first primer is a mixture of first primers, each primer comprising an oligo (dT) sequence and a 3′

end sequence of 1-5 nucleotides, and said mixture of first primers comprising primers having an A, a G, or a C nucleotide present in each position of said 3′

end sequence.

51. The method of claim 49 wherein each primer in said mixture of first primers further comprise at its 5′

end a selected sequence of 5-12 nucleotides.

52. The method of claim 50 wherein each primer in said mixture of first primers further comprises at its 5′

end a selected sequence of 5-12 nucleotides.

53. The method of claim 47 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence.

54. The method of claim 48 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence.

55. The method of claim 51 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence.

56. The method of claim 52 wherein said second primer is a mixture of second primers, each primer comprising at its 5′

end a selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C or a T nucleotide present in each position of said 3′

end sequence.

57. The method of claim 45 or 46 wherein said forward primer comprises said RNA polymerase promoter sequence.

58. The method of claim 45 or 46 wherein said reverse primer comprises said RNA polymerase promoter sequence.

59. The method of claim 55 wherein said forward primer comprises a 3′

end sequence of 8-12 nucleotides that is the same as the 5′

end sequence of 8-12 nucleotides of the second primer, and said reverse primer comprises a 3′

end sequence of 5-12 nucleotides that is the same as the 5′

end sequence of 5-12 nucleotides of the first primer.

60. The method of claim 56 wherein said forward primer comprises a 3′

end sequence of 8-12 nucleotides that is the same as the 5′

end sequence of 8-12 nucleotides of the second primer, and said reverse primer comprises a 3′

end sequence of 5-14 nucleotides that is the same as the 5′

end sequence of 5-12 nucleotides of the first primer.

61. The method of claim 55 wherein said reverse primer comprises said RNA polymerase promoter sequence.

62. The method of claim 59 wherein said reverse primer comprises said RNA polymerase promoter sequence.

63. The method of claim 45 wherein said sample contains total RNA or total mRNA from cells.

64. The method of claim 46 wherein said sample contains total RNA or total mRNA from cells.

65. The method of claim 59 or 60 wherein said sample contains total RNA or total mRNA from cells.

66. The method of claim 63 wherein said cells are yeast cells.

67. The method of claim 63 wherein said cells are mammalian cells.

68. The method of claim 45 wherein said sample contains total RNA from 1×

10⁶ cells or less.

69. The method of claim 46 wherein said sample contains total RNA from 1×

10⁶ cells or less.

70. The method of claim 59 or 60 wherein said sample contains total RNA from 1×

10⁶ cells or less.

71. The method of claim 45 wherein said first label is Cy3 and said second label is Cy5.

72. The method of claim 45 or 46 wherein said detecting or measuring steps (h) and (i) are carried out by a method comprising contacting said cRNA with an array containing one or more species of polynucleotide probe positioned at preselected sites on the array, under conditions conducive to hybridization;

and detecting any hybridization that occurs between said polynucleotide probes and said cRNA.

73. The method of claim 63 or 68 wherein said detecting or measuring steps (h) and (i) are carried out by a method comprising contacting said cRNA with an array containing one or more species of polynucleotide probe positioned at preselected sites on the array, under conditions conducive to hybridization;

and detecting any hybridization that occurs between said polynucleotide probes and said cRNA.

74. The method of claim 70 wherein said detecting or measuring steps (h) and (i) are carried out by a method comprising contacting said cRNA with an array containing one or more species of polynucleotide probe positioned at preselected sites on the array, under conditions conducive to hybridization;

and detecting any hybridization that occurs between said polynucleotide probes and said cRNA.

76. The method of claim 72 wherein the array comprises a support with at least one surface and at least 100 different polynucleotide probes, each different polynucleotide probe comprising a different nucleotide sequence and being attached to the surface of the support in a different, selected location on said surface.

83. The method of claim 45 or 46 wherein in steps (h) and (i), the steps of contacting the cRNA labeled with said first label with said polynucleotide probe, and contacting the cRNA labeled with said second label with said polynucleotide probe, are carried out concurrently.

84. The method of claim 59 or 60 wherein in steps (h) and (i), the steps of contacting the cRNA labeled with said first label with said polynucleotide probe, and contacting the cRNA labeled with said second label with said polynucleotide probe, are carried out concurrently.

85. The method of claim 65 wherein in steps (h) and (i), the steps of contacting the cRNA labeled with said first label with said polynucleotide probe, and contacting the cRNA labeled with said second label with said polynucleotide probe, are carried out concurrently.

86. The method of claim 45 wherein said first sample contains mRNAs from cells that are diseased and wherein said second sample contains mRNAs from cells that are not so diseased.

87. A kit comprising in one or more containers:

(a) a mixture of first primers, each first primer comprising a 3′

end sequence of 1-5 nucleotides, said mixture of first primers comprising primers having an A, a G, or a C nucleotide present in each position of said 3′

end sequence;

wherein each primer in said mixture of first primers further comprises at its 5′

end an identical selected sequence of 5-12 nucleotides, and optionally further comprises an oligo (dT) sequence situated between said 5′

end sequence and said 3′

end sequence;

(b) a mixture of second primers, each second primer comprising at its 5′

end an identical selected sequence of 8-12 nucleotides, and at its 3′

end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′

end sequence;

wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation; and

(c) a primer pair suitable for use in PCR, said primer pair comprising a forward primer and a reverse primer, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation.

88. The kit of claim 87 wherein the forward primer comprises said RNA polymerase promoter sequence.

89. The kit of claim 87 wherein said reverse primer comprises said RNA polymerase promoter sequence.

90. The kit of claim 87 wherein said forward primer comprises a 3′

end sequence of 8- 

nucleotides that is the same as the 5′

end sequence of 8-12 nucleotides of the second primer, and said reverse primer comprises a 3′

end sequence of 5-12 nucleotides that is the same as the 5′

end sequence of 5-12 nucleotides of the first primer.

91. The kit of claim 88 wherein said forward primer comprises a 3′

end sequence of 8-12 nucleotides that is the same as the 5′

end sequence of 8-12 nucleotides of the second primer, and said reverse primer comprises a 3′

end sequence of 5-12 nucleotides that is the same as the 5′

end sequence of 5-12 nucleotides of the first primer.

92. The kit of claim 87, which further comprises in a separate container an RNA polymerase specific to said RNA polymerase promoter.

93. The kit of claim 87 which further comprises a buffer for PCR.

94. The kit of claim 93 which further comprises a DNA polymerase.

95. The kit of claim 87, which further comprises:

(h) a set of directions for carrying out PCR; and

(i) a set of directions for carrying out transcribing of amplified double-stranded cDNA into cRNA.

96. The kit of claim 87 further comprising a control nucleic acid.

97. The kit of claim 87 further comprising a microarray.

98. The kit of claim 97 further comprising means for stimulating and detecting fluorescent light emissions from fluorescently labeled RNA.

99. The kit of claim 98 further comprising expression profile projection and analysis software capable of being loaded into the memory of a computer system.