Quantitative analysis methods on active electronic microarrays
First Claim
1. A method of providing an internal control for an individual test site in a nucleic acid hybridization reaction assay to determine the presence of at least one nucleic acid sequence of interest in at least one nucleic acid containing sample, wherein the nucleic acid hybridization assay is performed on an electronically controlled microarray comprising at least two test sites, the method comprising:
- (a) attaching a nucleic acid probe mix consisting of a first nucleic acid probe specific for a first internal control nucleic acid sequence known to be present in the sample, and a second nucleic acid probe specific for a second nucleic acid sequence of interest to a first test site on the electronically controlled microarray;
(b) attaching a mixed nucleic acid probe consisting of the first nucleic acid probe and a third nucleic acid probe specific for a third nucleic acid sequence of interest, wherein the third nucleic acid sequence of interest may be the same as or different than the second nucleic acid sequence of interest, to a second test site on the electronically controlled microarray;
(c) electronically hybridizing the sample nucleic acids from at least one sample to the nucleic acid probes on the first and second test sites;
(d) specifically detecting the extent of hybridization of the sample nucleic acids to the first nucleic acid probe at the first and second test sites;
(e) specifically detecting the extent of hybridization of the sample nucleic acids to the second and third nucleic acid probes at the first and second test sites;
(f) comparing the hybridization values obtained for the first nucleic acid probe at the first and second test sites to obtain a normalization factor; and
(g) normalizing the hybridization values obtained in (e) for the second and third probes using the normalization factor obtained in (f).
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Abstract
The present invention presents techniques useful in methods for gene expression monitoring, and other nucleic acid hybridization assays, that utilize microelectronic arrays to drive the transport and hybridization of nucleic acids. Particularly, methods for normalizing the signals of individual microlocations by the use of an internal control sequence probe are provided. These methods are particularly useful for hybridization assays in which a quantitative comparison of the hybridization of several different sequences at a plurality of microlocations is desired, such as in gene expression analyses.
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Citations
14 Claims
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1. A method of providing an internal control for an individual test site in a nucleic acid hybridization reaction assay to determine the presence of at least one nucleic acid sequence of interest in at least one nucleic acid containing sample, wherein the nucleic acid hybridization assay is performed on an electronically controlled microarray comprising at least two test sites, the method comprising:
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(a) attaching a nucleic acid probe mix consisting of a first nucleic acid probe specific for a first internal control nucleic acid sequence known to be present in the sample, and a second nucleic acid probe specific for a second nucleic acid sequence of interest to a first test site on the electronically controlled microarray;
(b) attaching a mixed nucleic acid probe consisting of the first nucleic acid probe and a third nucleic acid probe specific for a third nucleic acid sequence of interest, wherein the third nucleic acid sequence of interest may be the same as or different than the second nucleic acid sequence of interest, to a second test site on the electronically controlled microarray;
(c) electronically hybridizing the sample nucleic acids from at least one sample to the nucleic acid probes on the first and second test sites;
(d) specifically detecting the extent of hybridization of the sample nucleic acids to the first nucleic acid probe at the first and second test sites;
(e) specifically detecting the extent of hybridization of the sample nucleic acids to the second and third nucleic acid probes at the first and second test sites;
(f) comparing the hybridization values obtained for the first nucleic acid probe at the first and second test sites to obtain a normalization factor; and
(g) normalizing the hybridization values obtained in (e) for the second and third probes using the normalization factor obtained in (f). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification