Expression profiling using microarrays

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.

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.   
     
     
         2 . The method of  claim 1 , comprising introducing an identical barcode into each of the plurality of replicate nucleic acids of an expressed RNA sample. 
     
     
         3 . The method of  claim 1 , comprising introducing a different barcode into each of the replicate nucleic acids corresponding to a different polynucleotide sequence of the subset. 
     
     
         4 . The method of  claim 1 , wherein the replicate nucleic acids are produced by reverse transcription. 
     
     
         5 . The method of  claim 1 , wherein the replicate nucleic acids are produced by amplification. 
     
     
         6 . The method of  claim 5 , wherein the replicate nucleic acids are produced by selective amplification of the plurality of expressed RNA samples. 
     
     
         7 . The method of  claim 6 , wherein the amplified nucleic acids are produced by selective amplification by one or more method selected from the group consisting of: PCR, TMA, NASBA, and RCA. 
     
     
         8 . The method of  claim 7 , wherein the selective amplification is performed by PCR. 
     
     
         9 . The method of  claim 7 , wherein the selective amplification is performed in a multiplex reaction using a plurality of gene specific primers. 
     
     
         10 . The method of  claim 9 , wherein the gene specific primers further comprise a universal priming sequence. 
     
     
         11 . The method of  claim 10 , wherein the primer comprising the universal priming sequence further comprises a barcode sequence. 
     
     
         12 . The method of  claim 10 , wherein the primer comprising the universal priming sequence further comprises a detectable moiety. 
     
     
         13 . The method of  claim 12 , wherein the detectable moiety comprises a fluorescent label. 
     
     
         14 . The method of  claim 1 , comprising arraying the replicate nucleic acids by hybridizing them to an array of defined sequence probes. 
     
     
         15 . The method of  claim 1 , comprising arraying the replicate nucleic acids by hybridizing them to an array comprising polynucleotide sequences that hybridize to the at least one introduced barcode sequence. 
     
     
         16 . The method of  claim 1 , wherein the replicate nucleic acids are pooled for arraying. 
     
     
         17 . The method of  claim 6 , wherein the selective amplification amplifies between about 5 and about 100 polynucleotide sequences. 
     
     
         18 . The method of  claim 6 , wherein the selective amplification amplifies between about 10 and about 50 polynucleotide sequences. 
     
     
         19 . The method of  claim 6 , comprising amplifying each expressed RNA sample in two or more target specific amplification reactions and spatially arraying the resulting amplification products in two or more locations on an array. 
     
     
         20 . The method of  claim 1 , comprising introducing at least a second barcode sequence into the plurality of replicate nucleic acids. 
     
     
         21 . The method of  claim 1 , comprising detecting the at least one arrayed nucleic acid by hybridizing a defined sequence probe comprising a detectable moiety. 
     
     
         22 . The method of  claim 21 , comprising hybridizing a plurality of defined sequence probes, which probes each comprise a different polynucleotide sequence, and which probes are each capable of generating a different detectable signal, to the nucleic acid array. 
     
     
         23 . The method of  claim 1 , comprising detecting the at least one arrayed nucleic acid by hybridizing a barcode specific probe comprising a detectable moiety. 
     
     
         24 . The method of  claim 21  or  23 , wherein the detectable moiety comprises a fluorescent label. 
     
     
         25 . The method of  claim 24 , comprising hybridizing a plurality of probes, each comprising a different fluorescent label. 
     
     
         26 . The method of  claim 1 , comprising detecting the at least one arrayed nucleic acid by hybridizing a linking oligonucleotide, which linking nucleic acid comprises at least a first subsequence that hybridizes to a gene specific sequence and at least a second subsequence comprising a barcode sequence; and, hybridizing a probe comprising a detectable moiety to the barcode sequence. 
     
     
         27 . The method of  claim 1 , comprising obtaining the plurality of expressed RNA samples from each of a plurality of biological samples, each of which biological samples has been contacted with at least one member of a compound library. 
     
     
         28 . The method of  claim 1 , further comprising quantitating the detected signal. 
     
     
         29 . The method of  claim 28 , comprising comparing the quantitated signal to a control signal. 
     
     
         30 . The method of  claim 29 , wherein the quantitated signal is increased or decreased relative to the control signal. 
     
     
         31 . The method of  claim 30 , comprising detecting the quantitated signal that differs from a control signal by performing at least one statistical analysis. 
     
     
         32 . The method of  claim 1 , wherein the biological samples comprise one or more of: a tissue, a tissue extract, a primary cell isolate and cells grown in culture. 
     
     
         33 . The method of  claim 32 , wherein the biological samples comprise one or more cell lines. 
     
     
         34 . The method of  claim 32 , wherein each biological sample is contacted with a member of a compound library prior to collection of the expressed RNA sample. 
     
     
         35 . The method of  claim 33 , wherein each biological sample is contacted with a different member of the compound library. 
     
     
         36 . The method of  claim 33 , wherein expression of one or more genes in the one or more cell lines is artificially altered prior to treating with a member of a compound library using a procedure selected from the group consisting of: insertional mutagenesis, deletion of genomic DNA, targeted gene disruption, transcription blocking, introduction of a genomic or episomal vector, antisense DNA or RNA, ribozymes, iRNA, DNA binding oligonucleotides, and zinc finger proteins. 
     
     
         37 . The method of  claim 27 , wherein the biological samples comprise eukaryotic samples. 
     
     
         38 . The method of  claim 27 , wherein the biological samples comprise prokaryotic samples. 
     
     
         39 . The method of  claim 27 , wherein the compound library comprises one or more of: a compound collection library, a combinatorial chemical library, a scaffold-focused chemical library, a target focused chemical library, an antibody library, a biological library, a natural product library, an antisense agent library, an iRNA library, a siRNA library, a ribozyme library, a peptide library, and a combinatorial nucleic acid oligomer library. 
     
     
         40 . The method of  claim 1 , comprising obtaining expressed RNA samples from at least 500 biological samples. 
     
     
         41 . The method of  claim 1 , comprising obtaining expressed RNA samples from at least 1000 biological samples. 
     
     
         42 . The method of  claim 1 , comprising obtaining expressed RNA samples from at least 10,000 biological samples. 
     
     
         43 . The method of  claim 1 , comprising obtaining the one or more expressed RNA samples by isolating total cellular RNA. 
     
     
         44 . The method of  claim 1 , comprising obtaining the one or more expressed RNA samples by isolating messenger RNA (mRNA). 
     
     
         45 . The method of  claim 1 , comprising arraying a plurality of RNAs, cDNAs or amplified nucleic acids corresponding to the plurality of expressed RNA samples. 
     
     
         46 . The method of  claim 45 , comprising arraying a plurality of amplified nucleic acids corresponding to the plurality of expressed RNA samples, which amplified nucleic acids are produced by selective amplification of the plurality of expressed. RNA samples. 
     
     
         47 . The method of  claim 22 , comprising (i) hybridizing at least a first defined sequence probe and at least a second defined sequence probe, which first defined sequence probe hybridizes to a housekeeping gene and which at least second defined sequence probe hybridizes to a target sequence; (ii) quantitating the hybridization signals for the first and at least second defined sequence probes; and, (iii) determining the expression of the at least second defined sequence probe relative to the first defined sequence probe. 
     
     
         48 . The method of  claim 47 , wherein the nucleic acids corresponding to the expressed RNA samples are arrayed in two or more duplicate arrays, and each array is hybridized to the first defined sequence probe and the least a second defined sequence probe, wherein the first defined sequence probe is the same between the two or more duplicate arrays and the at least second defined sequence probe differs between the two or more duplicate arrays. 
     
     
         49 . The method of  claim 22 , wherein plurality of defined sequence probes comprises set of genes comprising disease related targets. 
     
     
         50 . The method of  claim 1 , comprising arraying the nucleic acids on a solid phase surface. 
     
     
         51 . The method of  claim 50 , comprising arraying the nucleic acids on a two dimensional solid phase surface. 
     
     
         52 . The method of  claim 50 , comprising arraying the nucleic acids on a plurality of solid phase surfaces. 
     
     
         53 . The method of  claim 52 , wherein the plurality of solid phase surfaces are selected from the group consisting of: beads, spheres and optical fibers. 
     
     
         54 . The method of  claim 50 , wherein the solid phase surface comprises a material selected from the group consisting of: glass, coated glass, silicon, porous silicon, nylon, ceramic and plastic. 
     
     
         55 . The method of  claim 22 , wherein the defined sequence probes comprise one or more synthetic, probes selected from the group consisting of: an oligonucleotide, a cDNA; an amplification product, and a restriction fragment. 
     
     
         56 . The method of  claim 22 , wherein the defined sequence probes capable of generating a detectable signal comprise one or more of: a fluorescent label, a chromophore, an electrophore, a radioactive nuclide, a chemically reactive moiety, an amplifiable signal element and a ligand capable of binding to an enzyme. 
     
     
         57 . The method of  claim 56 , wherein the amplifiable signal element is an oligonucleotide. 
     
     
         58 . The method of  claim 57 , wherein at least one of the plurality of defined sequence probes comprising an amplifiable signal element is detected by one or more of branched DNA amplification (BDA), rolling circle amplification (RCA), hybridization signal amplification method (HSAM), ramification amplification method (RAM) and a DNA dendrimer probe. 
     
     
         59 . The method of  claim 57 , wherein at least one of the plurality of defined sequence probes comprises an amplifiable signal element, which amplifiable signal element comprises a ligand which binds to a second amplifiable signal element. 
     
     
         60 . The method of  claim 56 , wherein the amplifiable signal element comprises an enzyme or a catalyst. 
     
     
         61 . The method of  claim 1 , further comprising amplifying at least one detectable signal prior to detecting the signal corresponding to the replicate nucleic acid. 
     
     
         62 . A kit comprising:
 (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.   
     
     
         63 . The kit of  claim 62 , further comprising a microarray comprising a set of nucleic acids, wherein each member of the set of nucleic acids is located at a different physical location within the array. 
     
     
         64 . The kit of  claim 62 , wherein each gene specific sequence hybridizes to a polynucleotide sequence in an expressed RNA sample. 
     
     
         65 . A method of determining a gene expression profile, comprising:
 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.   
     
     
         66 . The method of  claim 65 , wherein providing the RNA sample comprises obtaining RNA from a biological sample. 
     
     
         67 . The method of  claim 66 , wherein the biological sample comprises a cell culture. 
     
     
         68 . The method of  claim 66 , wherein the biological sample comprises a tissue sample from a patient. 
     
     
         69 . The method of  claim 65 , wherein providing the RNA sample further comprises providing a control RNA sequence. 
     
     
         70 . The method of  claim 65 , wherein amplifying the RNA sample comprises performing a global amplification of member ribonucleic acids. 
     
     
         71 . The method of  claim 65 , wherein amplifying the RNA sample comprises performing a selective amplification of a subset of member ribonucleic acids. 
     
     
         72 . The method of  claim 65 , wherein the at least one primer comprising the at least one universal priming, sequence further comprises a detectable moiety, thereby generating a set of detectable PCR products. 
     
     
         73 . The method of  claim 72 , wherein the detectable moiety comprises a fluorescent label. 
     
     
         74 . The method of  claim 73 , wherein the universal primer is labeled prior to generating the set of PCR products. 
     
     
         75 . The method of  claim 73 , wherein the universal primer is labeled after generating the set of PCR products. 
     
     
         76 . The method of  claim 65 , wherein the gene-specific primers further comprise at least one barcode sequence. 
     
     
         77 . The method of  claim 76 , wherein a first pair of gene-specific primers comprising a universal priming sequence further comprise a first bar code sequence, and wherein a second pair of gene-specific primers comprising a universal priming sequence further comprise a second barcode sequence. 
     
     
         78 . The method of  claim 65 , wherein amplifying the RNA sample further comprises pooling two or more sets of PCR products. 
     
     
         79 . The method of  claim 65 , wherein the array comprises a microarray. 
     
     
         80 . The method of  claim 65 , wherein the array comprises a dot blot array. 
     
     
         81 . The method of  claim 65 , wherein the array comprises an ordered array. 
     
     
         82 . The method of  claim 65 , wherein providing the array comprises providing a two-dimensional array of nucleic acids. 
     
     
         83 . The method of  claim 65 , wherein providing the array comprises providing a three-dimensional array of nucleic acids. 
     
     
         84 . The method of  claim 65 , wherein providing the RNA sample comprises providing a first RNA sample from a first biological source and a second RNA sample from a second biological source, and wherein hybridizing member PCR products to complementary member nucleic acids of the array comprises performing a competitive hybridization. 
     
     
         85 . The method of  claim 84 , wherein member PCR products generated from the first or the second RNA samples are labeled. 
     
     
         86 . The method of  claim 84 , wherein member PCR products generated from both the first and the second RNA samples are labeled. 
     
     
         87 . The method of  claim 65 , further comprising removing unbound PCR products from the array. 
     
     
         88 . The method of  claim 87 , wherein removing the unbound PCR products comprises washing the array with a low stringency buffer. 
     
     
         89 . The method of  claim 65 , wherein at least one primer comprising a universal priming sequence comprises a fluorescence label, and wherein detecting the hybridized member PCR product comprises determining an intensity of the fluorescence label. 
     
     
         90 . The method of  claim 65 , wherein detecting the hybridized member PCR product comprises detecting a radiolabel. 
     
     
         91 . The method of  claim 65 , wherein detecting the hybridized member PCR product further comprises quantitating a relative amount of the hybridized member PCR product. 
     
     
         92 . A method of determining a gene expression profile comprising:
 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.   
     
     
         93 . The method of  claim 92 , wherein the at least one universal primer is a labeled universal primer. 
     
     
         94 . The method of  claim 93 , wherein the labeled universal primer comprises a fluorescence label. 
     
     
         95 . The method of  claim 93 , wherein the labeled universal primer comprises a radiolabel. 
     
     
         96 . The method of  claim 92 , wherein hybridizing comprises co-hybridizing at least two sets of bar coded PCR products derived from at least two different biological samples to the array. 
     
     
         97 . A kit for determining the gene expression level of a selected set of genes, the kit comprising:
 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.   
     
     
         98 . The kit of  claim 97 , wherein the set of nucleic acids representing the selected set of genes comprises a plurality of nucleic acid sequences complementary to portions of the genes. 
     
     
         99 . The kit of  claim 97 , wherein the plurality of gene-specific primer pairs further comprise a third bar code sequence portion;
 and wherein the set of nucleic acids representing the selected set of genes comprises a plurality of nucleic acids complementary to the third bar code sequence portions of the gene-specific primer pairs.   
     
     
         100 . A kit for determining a diagnosis or prognosis of a disease, the kit comprising:
 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.   
     
     
         101 . The kit of  claim 100 , wherein the set of nucleic acids representing the selected set of genes comprises a plurality of nucleic acid sequences complementary to portions of the member genes. 
     
     
         102 . The kit of  claim 100 , wherein the plurality of gene-specific primer pairs further comprise a third bar code sequence portion;
 and wherein the set of nucleic acids representing the selected set of genes comprises a plurality of nucleic acids complementary to the third bar code sequence portions of the gene-specific primer pairs.