Expression monitoring by hybridization to high density oligonucleotide arrays

Abstract

The present invention provides methods for comparing and identifying differences in nucleic acid sequences using a plurality of sequence specific recognition reagents (i.e., probes comprising a nucleic acid complementary to a nucleic acid sequence in collections to be compared) bound to a solid surface.

1 . A method of monitoring RNA processing, said method comprising:
 (a) providing a pool of target nucleic acids comprising RNA transcripts of one or more genes, or nucleic acids derived therefrom using said RNA transcripts as templates;   (b) hybridizing said pool of target nucleic acids to an array of oligonucleotide probes immobilized on a surface, said array comprising more than 100 different oligonucleotides, at least some of which comprise control probes, wherein each different oligonucleotide is localized in a predetermined region of said surface, the density of said different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm 2 , and at least some of said oligonucleotide probes are complementary to said RNA transcripts or said nucleic acids derived therefrom using said RNA transcripts; and   (c) quantifying the hybridization of said nucleic acids to said array, wherein said quantification is proportional to the expression level of said genes.   
     
     
         2 . The method of  claim 1 , wherein said pool of target nucleic acids comprises nascent RNA transcripts. 
     
     
         3 . The method of  claim 2 , wherein said pool of target nucleic acids further comprises processing intermediates. 
     
     
         4 . The method of  claim 2 , wherein said pool of target nucleic acids further comprises mature mRNA. 
     
     
         5 . The method of  claim 3 , wherein said pool of target nucleic acids further comprises mature mRNA. 
     
     
         6 . The method of  claim 1 , wherein said pool of target nucleic acids comprises alternatively spliced mRNA transcripts. 
     
     
         7 . The method of  claim 1 , wherein said array of oligonucleotides further comprises mismatch control probes. 
     
     
         8 . The method of  claim 7 , wherein said quantifying comprises calculating the difference in hybridization signal intensity between each of said oligonucleotide probes and its corresponding mismatch control probe. 
     
     
         9 . The method of  claim 8 , wherein said quantifying comprises calculating the average difference in hybridization signal intensity between each of said oligonucleotide probes and its corresponding mismatch control probe for each gene. 
     
     
         10 . The method of  claim 1 , wherein hybridization and quantification is accomplished in under 48 hours. 
     
     
         11 . The method of  claim 1 , wherein said multiplicity of genes is 100 genes or more. 
     
     
         12 . The method of  claim 1 , wherein for each gene, said array comprises at least 10 different oligonucleotide probes complementary to subsequences of that gene. 
     
     
         13 . The method of  claim 1 , wherein said hybridization is performed with a fluid volume of about 250 μl or less. 
     
     
         14 . The method of  claim 1 , wherein said quantifying comprises detecting a hybridization signal that is proportional to the concentration of said RNA in said nucleic acid sample. 
     
     
         15 . The method of  claim 1 , wherein said oligonucleotides are from 5 to about 50 nucleotides in length. 
     
     
         16 . The method of  claim 1 , wherein said oligonucleotides are synthesized by light-directed polymer synthesis. 
     
     
         17 . The method of  claim 1 , wherein said control probes comprise sequences from constitutively expressed control genes. 
     
     
         18 . The method of  claim 12 , wherein said control genes are selected from the group consisting of β-actin, GAPDH, and the transferrin receptor. 
     
     
         19 . The method of  claim 1 , wherein said hybridization comprises a hybridization at low stringency of 30° C. to 50° C. and 6×SSPE-T or lower and a wash at higher stringency. 
     
     
         20 . The method of  claim 1 , wherein said pool of target nucleic acids is a pool of mRNAs. 
     
     
         21 . The method of  claim 1 , wherein said pool of target nucleic acids is a pool of RNAs in vitro transcribed from a pool of cDNAs. 
     
     
         22 . The method of  claim 1 , wherein said pool of target nucleic acids is amplified from a biological sample by an in vivo or an in vitro method. 
     
     
         23 . The method of  claim 1 , wherein said pool of target nucleic acids comprises fluorescently labeled nucleic acids. 
     
     
         24 . The method of  claim 1 , wherein said detecting comprises quantifying fluorescence of a label on said hybridized nucleic acids at a spatial resolution of 100 μm or higher. 
     
     
         25 . The method of  claim 24 , wherein said quantification is by means of a scanning confocal fluorescence microscope. 
     
     
         26 . The method of  claim 1 , wherein said providing comprises:
 (i) hybridizing a pool of RNAs with a pool of oligonucleotides having the same sequences as said oligonucleotide probes to form a pool of hybridized nucleic acids;   (ii) treating said pool of hybridized nucleic acids with RNase A, thereby digesting single stranded nucleic acid sequences and leaving intact the hybridized double stranded regions;   (iii) denaturing the hybridized double-stranded regions and removing said oligonucleotides thereby leaving a pool of RNAs enhanced for those RNAs complementary to the oligonucleotide probes in said high density array.   
     
     
         27 . The method of  claim 1 , wherein said providing comprises:
 (a) hybridizing a pool of RNAs with paired target specific oligonucleotides wherein said paired target specific oligonucleotides are complementary to regions on either side of a subsequence, said subsequence being complementary to at least one of said oligonucleotide probes in said array;   (b) treating said pool of nucleic acids with RNase H to digest the hybridized (double stranded) nucleic acid sequences; and   (c) isolating the remaining nucleic acid sequences having a length about equivalent to said subsequence.   
     
     
         28 . The method of  claim 1 , wherein said providing comprises:
 (a) hybridizing a pool of poly A +  mRNAs with oligonucleotides that hybridize specifically with preselected mRNA target messages;   (b) treating said pool of nucleic acids with RNase H to digest the hybridized (double stranded) nucleic acid sequences thereby separating the coding sequence from the polyA +  tail; and   (c) isolating or amplifying the polyA +  RNA in said pool.   
     
     
         29 . The method of  claim 1 , wherein at least some of said oligonucleotides are up to about 500 nucleotides in length. 
     
     
         30 . The method of  claim 1 , wherein at least some of said oligonucleotides are greater than 10 nucleotides in length. 
     
     
         31 . The method of  claim 1 , wherein at least some of the oligonucleotides are chosen based on sequence information from at least one public database. 
     
     
         32 . The method of  claim 1 , wherein said surface is glass. 
     
     
         33 . The method of  claim 1 , wherein said surface is a chip. 
     
     
         34 . The method as recited in  claim 1  wherein said probes are provided by synthesis of RNA or DNA on the solid surface. 
     
     
         35 . The method as recited in  claim 1  wherein said probes are delivered to the surface through flow channels on the surface of the substrate. 
     
     
         36 . The method as recited in  claim 1  wherein said probes are delivered to the surface by spotting on predefined regions. 
     
     
         37 . The method of  claim 1  wherein at least some of said oligonucleotide probes are specific for splice variants associated with a disease state. 
     
     
         38 . The method of  claim 37  wherein at least some of said oligonucleotide probes are specific for splice variants associated with malignant transformation. 
     
     
         39 . The method of  claim 37  wherein at least some of said oligonucleotide probes are specific for splice variants associated with apoptosis. 
     
     
         40 . The method of  claim 1  wherein said method is used in toxicology. 
     
     
         41 . The method of  claim 1  wherein said method is used in drug discovery. 
     
     
         42 . The method of  claim 1  wherein said method is used in medical diagnostics. 
     
     
         43 . A device for performing the method of  claim 1  comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to nascent RNA transcripts. 
     
     
         44 . A device for performing the method of  claim 1  comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to RNA processing intermediates. 
     
     
         45 . A device for performing the method of  claim 1  comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to mature mRNA. 
     
     
         46 . A device for performing the method of  claim 1  comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to alternatively spliced mRNA transcripts.