Systems and methods for correcting error in biological response signal data

• US 20050130215A1
• Filed: 01/24/2005
• Published: 06/16/2005
• Est. Priority Date: 12/28/1998
• Status: Abandoned Application

• Current Assignee: Microsoft Technology Licensing LLC (Microsoft Corporation)
• Original Assignee: Microsoft Technology Licensing LLC (Microsoft Corporation)
• Inventors: Stoughton, Roland, Dai, Hongyue
• Application Number:
  US11/042,653
• Publication Number:
  US 20050130215A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

A method for fluorophore bias removal in microarray experiments in which the fluorophores used in microarray experiment pairs are reversed. Further, a method for calculating the individual errors associated with each measurement made in nominally repeated microarray experiments. This error measurement is optionally coupled with rank based methods in order to determine a probability that a cellular constituent is up or down regulated in response to a perturbation. Finally, a method for determining the confidence in the weighted average of the expression level of a cellular constituent in nominally repeated microarray experiments.

Claims

1. A method of fluorophore bias removal comprising the steps of:

(a) labeling a first pool of genetic matter, derived from a biological system representing a baseline state, with a first fluorophore to obtain a first pool of fluorophore-labeled genetic matter;

(b) labeling a second pool of genetic matter, derived from a biological system representing a perturbed state, with a second fluorophore to obtain a second pool of fluorophore-labeled genetic matter;

(c) labeling a third pool of genetic matter, derived from said biological system representing said baseline state, with said second fluorophore to obtain a third pool of fluorophore-labeled genetic matter;

(d) labeling a fourth pool of genetic matter, derived from said biological system representing said perturbed state, with said first fluorophore to obtain a fourth pool of fluorophore-labeled genetic matter;

(e) contacting said first pool of fluorophore-labeled genetic matter and said second pool of fluorophore-labeled genetic matter with a first microarray under conditions such that hybridization can occur, respectively detecting a first and second flourescent emission signal at each of a plurality of discrete loci on the first microarray from said first and second pool of fluorophore-labeled genetic matter that is bound to said first microarray under said conditions, and determining a first color ratio between said first flourescent emission signal and said second flourescent emission signal;

(f) contacting said third pool of fluorophore-labeled genetic matter and said fourth pool of fluorophore-labeled genetic matter with a second microarray under conditions such that hybridization can occur, respectively detecting a third and fourth flourescent emission signal at each of a plurality of discrete loci on the second microarray from said third and fourth pool of fluorophore-labeled genetic matter that is bound to said second microarray under said conditions, and determining a second color ratio between said third flourescent emission signal and said fourth flourescent emission signal; and

(g) computing an average color ratio by averaging said first color ratio and said second color ratio;

wherein;

said first microarray and said second microarray are similar to each other, exact replicas of each other, or are identical; and

said first color ratio and said second color ratio are determined for the same individual component of genetic matter, thereby removing fluorophore bias.

2-42. -42. (canceled)

43. A method of estimating error of a signal measured from a probe spot in a microarray experiment, the method comprising:

(A) accounting for an additive error in said probe spot; and

(B) accounting for a multiplicative error in said probe spot, wherein

said accounting for said additive error (A) is determined by a variance in a first measure of intensity of said probe spot and a variance in a second measure of intensity of said probe spot; and

said accounting for said multiplicative error (B) is determined by said first measure of intensity of said probe spot and said second measure of intensity of said probe spot.

44. The method of claim 43 wherein said first measure of intensity corresponds to an intensity of a first fluorophore at said probe spot in a first microarray and wherein said second measure of intensity corresponds to an intensity of a second fluorophore at said probe spot in said first microarray.

45. The method of claim 43 wherein said first measure of intensity corresponds to an intensity of a fluorophore at said probe spot in a first microarray and wherein said second measure of intensity corresponds to an intensity of said fluorophore at said probe spot in a second microarray, wherein said probe spot in said first microarray corresponds to said probe spot in said second microarray.

46. The method of claim 45 wherein said first microarray represents abundance levels of cellular constituents of a biological system when said biological system is in a base line state, and said second microarray represents abundance levels of cellular constituents of the biological system when said biological system is in a perturbed state.

47. The method of claim 46 wherein said biological system is a cell line, a cell culture, or a tissue sample obtained from a subject.

48. The method of claim 46 wherein said biological system is a Homo sapien.

49. The method of claim 46 wherein said perturbed state is achieved by exposing said biological system to a drug candidate or a pharmacological agent.

50. The method of claim 46 wherein said perturbed state is achieved by an introduction of an exogenous gene into the biological system or by a deletion of a gene from the biological system.

51. The method of claim 46 wherein said perturbed state is achieved by a change in a culture condition of the biological system.

52. The method of claim 43, wherein said additive error comprises:

σ

_X²+σ

_Y²

wherein

σ

_X² is said variance in said first measure of intensity; and

σ

_Y² is said variance in said second measure of intensity.

53. The method of claim 43, wherein said multiplicative error comprises:

f²(X²+Y²)

wherein

X is the first measure of intensity of said probe spot;

Y is the second measure of intensity of said probe spot; and

f is a fractional multiplicative error.

54. The method of claim 53, the method further comprising:

deriving a value for said fractional multiplicative error f by fitting;

{square root}{square root over (σ

_X²+σ

_Y²+f²(X²+Y²))}

to a plurality of probe spots on a microarray, said plurality of probe spots comprising said probe spot, wherein

σ

_X² is said variance in said first measure of intensity; and

σ

_Y² is said variance in said second measure of intensity.

55. A computer program product for use in conjunction with a computer system, the computer program product comprising a computer readable storage medium and a computer program mechanism embedded therein, wherein

the computer program mechanism is for estimating error of a signal measured from a probe spot in a microarray experiment, the computer program mechanism comprising:

instructions for accounting for an additive error in said probe spot; and

instructions for accounting for a multiplicative error in said probe spot, wherein

said accounting for said additive error is determined by a variance in a first measure of intensity of said probe spot and a variance in a second measure of intensity of said probe spot; and

said accounting for said multiplicative error is determined by said first measure of intensity of said probe spot and said second measure of intensity of said probe spot.

56. The computer program product claim 55 wherein said first measure of intensity corresponds to an intensity of a first fluorophore at said probe spot in a first microarray and wherein said second measure of intensity corresponds to an intensity of a second fluorophore at said probe spot in said first microarray.

57. The computer program product of claim 55 wherein said first measure of intensity corresponds to an intensity of a fluorophore at said probe spot in a first microarray and wherein said second measure of intensity corresponds to an intensity of said fluorophore at said probe spot in a second microarray, wherein said probe spot in said first microarray corresponds to said probe spot in said second microarray.

58. The computer program product of claim 57 wherein said first microarray represents abundance levels of cellular constituents of a biological system when said biological system is in a base line state, and said second microarray represents abundance levels of cellular constituents of the biological system when said biological system is in a perturbed state.

59. The computer program product of claim 58 wherein said biological system is a cell line, a cell culture, or a tissue sample obtained from a subject.

60. The computer program product of claim 58 wherein said biological system is a Homo sapien.

61. The computer program product of claim 58 wherein said perturbed state is achieved by exposing said biological system to a drug candidate or a pharmacological agent.

62. The computer program product of claim 58 wherein said perturbed state is achieved by an introduction of an exogenous gene into the biological system or by a deletion of a gene from the biological system.

63. The computer program product of claim 58 wherein said perturbed state is achieved by a change in a culture condition of the biological system.

64. The computer program product of claim 55, wherein said additive error comprises:

σ

_X²+σ

_Y²

wherein

σ

_X² is said variance in said first measure of intensity; and

σ

_Y² is said variance in said second measure of intensity.

65. The computer program product of claim 55, wherein said multiplicative error comprises:

f²(X²+Y²)

wherein

X is the first measure of intensity of said probe spot;

Y is the second measure of intensity of said probe spot; and

f is a fractional multiplicative error.

66. The computer program product of claim 65, the computer program mechanism further comprising:

instructions for deriving a value for said fractional multiplicative error f by fitting;

{square root}{square root over (σ

_X²+σ

_Y²+f²(X²+Y²))}

to a plurality of probe spots on a microarray, said plurality of probe spots comprising said probe spot, wherein

σ

_X² is said variance in said first measure of intensity; and

σ

_Y² is said variance in said second measure of intensity.

67. A computer system for estimating error of a signal measured from a probe spot in a microarray experiment, the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor, wherein the one or more programs cause the processor to perform a method comprising:

accounting for an additive error in said probe spot; and

accounting for a multiplicative error in said probe spot, wherein

said accounting for said additive error is determined by a variance in a first measure of intensity of said probe spot and a variance in a second measure of intensity of said probe spot; and

said accounting for said multiplicative error is determined by said first measure of intensity of said probe spot and said second measure of intensity of said probe spot.

68. A method of estimating error of a signal from a probe spot in a two-fluorophore microarray experiment, the method comprising:

empirically fitting a value f such that the expression;

{square root}{square root over (σ

_Xi²+σ

_Yi²+f²(X_i²+Y_i²))}

fits an x-y plot for a plurality of probe spots in said two-fluorophore microarray experiment, wherein said plurality of probe spots comprises said probe spot, and wherein

a first axis of said x-y plot represents, for each respective probe spot in said plurality of probe spots, the mean of (i) a measure of intensity of a first fluorophore for said respective probe spot, and (ii) a measure of intensity of a second fluorophore for said respective probe spot;

a second axis of said x-y plot represents a ratio, for each respective probe spot in said plurality of probe spots, between (iii) a measure of intensity of said first fluorophore for said respective probe spot and (iv) a measure of intensity of said second fluorophore for said respective probe spot;

X_i² is a measure of intensity of said first fluorophore in an i^th probe spot in said plurality of probe spots;

Y_i² is a measure of intensity of said second fluorophore in said i^th probe spot;

σ

_Xi² is a variance in X_i² that represents an additive error of X_i²; and

σ

_Yi² is a variance in Y_i² that represents an additive error of Y_i².

69. The method of claim 68 wherein X_i² is measured from said probe spot in a first microarray and wherein Y_i² is measured from said probe spot in said first microarray.

70. The method of claim 68 wherein X_i² is measured from said probe spot in a first microarray and wherein Y_i² is measured from said probe spot in a second microarray, wherein said probe spot in said first microarray corresponds to said probe spot in said second microarray.

71. The method of claim 70 wherein said first microarray represents abundance levels of cellular constituents of a biological system when said biological system is in a base line state, and said second microarray represents abundance levels of cellular constituents of the biological system when said biological system is in a perturbed state.

72. The method of claim 71 wherein said biological system is a cell line, a cell culture, or a tissue sample obtained from a subject.

73. The method of claim 71 wherein said biological system is a Homo sapien.

74. The method of claim 71 wherein said perturbed state is achieved by exposing said biological system to a drug candidate or a pharmacological agent.

75. The method of claim 71 wherein said perturbed state is achieved by an introduction of an exogenous gene into the biological system or by a deletion of a gene from the biological system.

76. The method of claim 71 wherein said perturbed state is achieved by a change in a culture condition of the biological system.

77. A computer program product for use in conjunction with a computer system, the computer program product comprising a computer readable storage medium and a computer program mechanism embedded therein, wherein

the computer program mechanism is for estimating error of a probe spot in a two-fluorophore microarray experiment, the computer program mechanism comprising instructions for empirically fitting a value f such that the expression:

{square root}{square root over (σ

_Xi²+σ

_Yi²+f²(X_i²+Y_i²))}

fits an x-y plot for a plurality of probe spots in said two-fluorophore microarray experiment, wherein said plurality of probe spots comprises said probe spot, and wherein

a first axis of said x-y plot represents, for each respective probe spot in said plurality of probe spots, the mean of (i) a measure of intensity of a first fluorophore for said respective probe spot, and (ii) a measure of intensity of a second fluorophore for said respective probe spot;

a second axis of said x-y plot represents a ratio, for each respective probe spot in said plurality of probe spots, between (iii) a measure of intensity of said first fluorophore for said respective probe spot and (iv) a measure of intensity of said second fluorophore for said respective probe spot;

X_i² is a measure of intensity of said first fluorophore in an i^th probe spot in said plurality of probe spots;

Y_i² is a measure of intensity of said second fluorophore in said i^th probe spot;

σ

_Xi² is a variance in X_i² that represents an additive error of X_i²; and

σ

_Yi² is a variance in Y_i² that represents an additive error of Y_i².

78. A computer system for estimating error of a probe spot in a two-fluorophore microarray experiment, the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor, wherein the one or more programs cause the processor to empirically fit a value f such that the expression:

{square root}{square root over (σ

_Xi²+σ

_Yi²+f²(X_i²+Y_i²))}

fits an x-y plot for a plurality of probe spots in said two-fluorophore microarray experiment, wherein said plurality of probe spots comprises said probe spot, and wherein

a first axis of said x-y plot represents, for each respective probe spot in said plurality of probe spots, the mean of (i) a measure of intensity of a first fluorophore for said respective probe spot, and (ii) a measure of intensity of a second fluorophore for said respective probe spot;

a second axis of said x-y plot represents a ratio, for each respective probe spot in said plurality of probe spots, between (iii) a measure of intensity of said first fluorophore for said respective probe spot and (iv) a measure of intensity of said second fluorophore for said respective probe spot;

X_i² is a measure of intensity of said first fluorophore in an i^th probe spot in said plurality of probe spots;

Y_i² is a measure of intensity of said second fluorophore in said i^th probe spot;

σ

_Xi² is a variance in X_i² that represents an additive error of X_i²; and

σ

_Yi² is a variance in Y_i² that represents an additive error of Y_i².