Expression monitoring by hybridization to high density oligonucleotide arrays
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.