Expression profiling using microarrays
First Claim
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1. A method for simultaneously detecting a plurality of expression products from a plurality of biological samples, the method comprising:
- (a) obtaining a plurality of expressed RNA sample each comprising a plurality of polynucleotide sequences from each of a plurality of biological samples;
(b) introducing at least one barcode sequence into a plurality of replicate nucleic acids corresponding to at least a subset of the plurality of polynucleotide sequences of the expressed RNA samples;
(c) arraying the plurality of replicate nucleic acids to produce a nucleic acid array; and
,(d) detecting a plurality of signals corresponding to arrayed replicate nucleic acids.
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Abstract
The invention provides novel compositions and methods for the analysis of gene expression (e.g., expression profiling) using microarray-based technology. In some embodiments of the invention, the novel methods use gene-specific as well as universal amplification primers during sample preparation, and the methods permit the simultaneous analysis of multiple samples on the same microarray. Furthermore, some embodiments of the invention incorporate barcode sequences into the amplified products, thereby permitting the use of generic arrays and generic labeled probes.
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Citations
102 Claims
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1. A method for simultaneously detecting a plurality of expression products from a plurality of biological samples, the method comprising:
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(a) obtaining a plurality of expressed RNA sample each comprising a plurality of polynucleotide sequences from each of a plurality of biological samples; (b) introducing at least one barcode sequence into a plurality of replicate nucleic acids corresponding to at least a subset of the plurality of polynucleotide sequences of the expressed RNA samples; (c) arraying the plurality of replicate nucleic acids to produce a nucleic acid array; and
,(d) detecting a plurality of signals corresponding to arrayed replicate nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)
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62. A kit comprising:
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(a) a plurality of chimeric primers, which chimeric primers each comprise (i) a subsequence comprising a gene specific sequence;
(ii) a subsequence comprising a barcode sequence; and
(iii) a subsequence comprising a universal priming sequence; and
,(b) at least one universal primer that hybridizes to the universal priming sequence. - View Dependent Claims (63, 64)
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65. A method of determining a gene expression profile, comprising:
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a) providing an RNA sample; b) selectively amplifying a subset of members of the RNA sample by a reverse transcriptase polymerase chain reaction (rtPCR) and generating a set of PCR products, wherein the rtPCR is performed with a reaction mixture comprising at least one pair of gene-specific primers, which gene specific primers further comprise at least one universal priming sequence; c) providing an array comprising a set of nucleic acid members corresponding to a plurality of gene expression products, wherein the nucleic acid members of the set are positioned at discreet physical locations within the array, and wherein at least one member of the set is complementary to at least a portion of a member of the set of PCR products; d) hybridizing member PCR products to complementary member nucleic acids of the array; and
,e) detecting a hybridized member PCR product at a discreet physical location in the array, thereby determining a gene expression profile. - View Dependent Claims (66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91)
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92. A method of determining a gene expression profile comprising:
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obtaining RNA from one or more biological samples; amplifying the RNA by rtPCR using at least one universal primer and at least one pair of chimeric gene-specific bar coded universal primers, and generating a set of bar coded PCR products; providing an array comprising a set of nucleic acids representing a plurality of gene expression products, wherein members of the set of nucleic acids are positioned at discreet physical locations within the array, and wherein at least one member nucleic acid is complementary to a bar code sequence of the set of bar coded PCR products; hybridizing members of the set of bar coded PCR products to complementary member nucleic acids of the array; washing the array and removing unbound bar coded PCR products; and detecting and quantitating an amount of bar coded PCR product hybridized to a selected location within the array, thereby determining the gene expression profile. - View Dependent Claims (93, 94, 95, 96)
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97. A kit for determining the gene expression level of a selected set of genes, the kit comprising:
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a) an array comprising a set of nucleic acids representing a selected set of genes, wherein members of the set of nucleic acids are discreetly located at different physical sites within the array; b) at least one universal primer; and c) a plurality of gene-specific primer pairs, wherein member primers comprise a first sequence portion complementary to the universal primer and a second sequence portion complementary to a sequence of a member of the selected set of genes. - View Dependent Claims (98, 99)
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100. A kit for determining a diagnosis or prognosis of a disease, the kit comprising:
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a) an array comprising a set of nucleic acids representing a selected set of genes associated with the disease, wherein members of the set of nucleic acids are discreetly located at different physical sites within the array; b) at least one universal primer; and c) a plurality of gene-specific primer pairs, wherein member primers comprise a first sequence portion complementary to the universal primer and a second sequence portion complementary to a sequence of a member of the selected set of genes. - View Dependent Claims (101, 102)
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Specification