Expression monitoring by hybridization to high density oligonucleotide arrays

• US 20050158746A1
• Filed: 11/23/2004
• Published: 07/21/2005
• Est. Priority Date: 09/15/1995
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lockhart, David J., Gingeras, Thomas R., Brown, Eugene L., Chee, Mark, Wong, Gordon G.
• Application Number:
  US10/998,518
• Publication Number:
  US 20050158746A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1475 using image analysis for extracting features of the particle

Abstract

This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm², and the olignucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Claims

1. A method of simultaneously monitoring the expression of a multiplicity of genes, said method comprising:

(a) providing a pool of target nucleic acids comprising RNA transcripts of one or more of said genes, or nucleic acids derived from said RNA transcripts;

(b) hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on a surface, said array comprising more than 100 different oligonucleotides 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.sup.2, and said olignucleotide probes are complementary to said RNA transcripts or said nucleic acids derived from said RNA transcripts; and

(c) quantifying the hybridization of said nucleic acids to said array.

2. The method of claim 1, wherein the concentration of said RNA transcripts in said pool, or nucleic acids derived from said RNA transcripts, is proportional to the expression levels of said genes.

3. The method of claim 1, wherein said array of oligonucleotides further comprises mismatch control probes.

4. The method of claim 3, wherein said quantifying comprises calculating the difference in hybridization signal intensity between each of said oligonucleotide probes and its corresponding mismatch control probe.

5. The method of claim 4, 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 .mu.l 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 array comprises oligonucleotide sequences from constitutively expressed control genes.

18. The method of claim 17, wherein said control genes are selected from the group consisting of .beta.-actin, GAPDH, and the transferrin receptor.

19. The method of claim 1, wherein said hybridization comprises a hybridization at low stringency of 30.degree. C. to 50.degree. C. and 6.times.SSPE-T or lower and a wash at higher stringency.

20. The method of claim 1, wherein said pool of nucleic acids is a pool of mRNAs.

21. -54. (canceled)