Expression profiling using microarrays

• US 7,824,856 B2
• Filed: 09/10/2004
• Issued: 11/02/2010
• Est. Priority Date: 09/10/2003
• Status: Active Grant

• Current Assignee: Altheadx, Inc.
• Original Assignee: Althea Technologies Inc. (Ajinomoto Co., Inc.)
• Inventors: Monforte, Joseph
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US10/938,130
• Publication Number:
  US 20050170373A1
• Time in Patent Office: 2,244 Days
• Field of Search: None
• US Class Current: 435/6.14
• 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 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

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.

Claims

1. A method of determining two or more gene expression profiles comprising:

obtaining RNA from two or more biological samples;

amplifying the RNA from each of the two or more biological samples by rtPCR using at least one universal primer and at least one pair of chimeric gene-specific bar coded universal primers having a first sequence portion complementary to the universal primer, a second sequence portion complementary to a gene of interest, and a third sequence portion comprising a bar code sequence, thereby generating two or more sets 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 the bar code sequence present in the two or more sets of bar coded PCR products;

hybridizing members of the two or more sets of bar coded PCR products to the array, wherein the third sequence portion comprising the barcode sequence hybridizes to the complementary member nucleic acid 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 profiles for each of the two or more biological samples.

2. The method of claim 1, wherein the at least one universal primer is a labeled universal primer.

3. The method of claim 2, wherein the labeled universal primer comprises a fluorescence label.

4. The method of claim 2, wherein the labeled universal primer comprises a radiolabel.