Normalisation of microarray data based on hybridisation with an internal reference
First Claim
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1. A method for the identification of an analyte in a biological sample comprising the steps of:
- (a) providing a microarray comprising a substrate with predefined regions wherein each binding substance immobilized at a predefined region onto said substrate comprises a predetermined amount of receptor and a predetermined amount of an internal reference, (b) providing a reporter molecule that binds selectively to said internal reference, (c) adding said reporter molecule to said biological sample, (d) contacting said biological sample comprising said reporter molecule with said microarray under conditions that allow binding to take place between said receptor and said analyte, and between said internal reference and said reporter molecule, (e) determining the signal of said reporter molecule binding to said internal reference, (f) determining the signal of said analyte binding to said receptor, and (g) normalising said signal of step (f) for said signal of step (e), whereby said analyte is identified.
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Abstract
The invention relates to methods and corresponding arrays especially suited to correct for signal errors due to variations in sample preparation. Methods and compositions for performing quantitative array-based assays are provided. In the subject methods, both a reporter and an analyte is employed, where the reporter is characterized by binding selectively to an internal reference present on the array, i.e. at least a subset of, if not all of, the spots present on the array employed in the method contain an internal reference which can be bound by reporter.
27 Citations
34 Claims
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1. A method for the identification of an analyte in a biological sample comprising the steps of:
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(a) providing a microarray comprising a substrate with predefined regions wherein each binding substance immobilized at a predefined region onto said substrate comprises a predetermined amount of receptor and a predetermined amount of an internal reference, (b) providing a reporter molecule that binds selectively to said internal reference, (c) adding said reporter molecule to said biological sample, (d) contacting said biological sample comprising said reporter molecule with said microarray under conditions that allow binding to take place between said receptor and said analyte, and between said internal reference and said reporter molecule, (e) determining the signal of said reporter molecule binding to said internal reference, (f) determining the signal of said analyte binding to said receptor, and (g) normalising said signal of step (f) for said signal of step (e), whereby said analyte is identified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 15, 16, 17, 18, 19, 29, 32, 33, 34)
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10. A method for the normalization of a microarray comprising the steps of:
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(a) immobilizing onto said array a binding substance comprising a receptor and a predetermined amount of an internal reference, and (b) determining the signal generated by said internal reference by means of a reporter molecule which selectively binds to said internal reference. - View Dependent Claims (11, 12, 13, 14)
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20-23. -23. (canceled)
- 24. Microarray comprising a substrate with predefined regions, wherein each binding substance immobilized at a predefined region of said substrate comprises a receptor and a predetermined amount of an internal reference, wherein the signal generated by said internal reference is determined by means of a reporter molecule and wherein said reporter molecule selectively binds to said internal reference.
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26-28. -28. (canceled)
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30-31. -31. (canceled)
Specification