Statistical combining of cell expression profiles
First Claim
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, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray;
(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, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(g) computing an average color ratio by averaging said first color ratio and said second color ratio.
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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.
58 Citations
49 Claims
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1. A method of fluorophore bias removal comprising the steps of:
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(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, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray;
(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, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(g) computing an average color ratio by averaging said first color ratio and said second color ratio.
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2-42. -42. (canceled)
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43. 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 computes an average color ratio by averaging a first color ratio and a second color ratio, said first color ratio and said second color ratio having been determined by a method comprising: -
(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 fluorescent 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 becomes bound to said second microarray under said conditions, and determining said first color ratio between said first fluorescent emission signal and said second fluorescent 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 fluorescent 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 becomes bound to said second microarray under said conditions, and determining said second color ratio between said third fluorescent emission signal and said fourth fluorescent emission signal;
wherein;
said first microarray and said second microarray are similar to each other, exact replicas of each other, or are identical;
said first color ratio and said second color ratio are determined for the same individual component of genetic matter; and
said first fluorophore and said second fluorophore are different. - View Dependent Claims (44, 45, 46, 47, 48, 49)
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Specification