Gene expression profiling from FFPE samples

• US 20040072305A1
• Filed: 12/23/2002
• Published: 04/15/2004
• Est. Priority Date: 10/11/2002
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: BioTheranostics, Inc. (Hologic Incorporated)
• Inventors: Erlander, Mark G., Salunga, Ranelle C.
• Granted Patent: US 7,364,846 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation

Abstract

Methods and compositions relating to the generation and use of gene expression data from tissue samples that have been fixed and embedded are provided. The data can electronically stored and implemented as well as used to augment diagnosis and treatment of diseases.

Claims

1. A method of synthesizing cDNA from the polyadenylated RNA from cells of an FFPE sample comprising

a) extracting said RNA from said cells;

and

b) contacting said extracted RNA with a primer comprising an oligo dT sequence under conditions that result in the synthesis of a first cDNA strand complementary to said extracted RNA.

2. The method of claim 1 further comprising heating said RNA extracted from said cells at about 70°

C. before contacting said RNA with said primer.

3. The method of claim 1 wherein said extracting of RNA from said cells comprises

obtaining said cells from said sample;

digesting said cells with proteinase K to produce digested material;

contacted the digested material with a guanidinium containing compound to produce a mixture;

contacting the mixture with a silica matrix to permit RNA binding to the matrix; and

eluting the bound RNA after removal of unbound matter.

4. The method of claim 2 wherein said heating is for about 3 to about 8 hours.

5. The method of claim 4 wherein said heating is for about 3 hours.

6. The method of claim 1 further comprising synthesis of the second cDNA strand by use of random primers.

7. The method of claim 1 further comprising synthesis of the second cDNA strand in the absence of random primers.

8. The method of claim 1 wherein said primer is operably linked to a promoter sequence.

9. The method of claim 6 wherein said random primers are hexamers, heptamers, octamers, or nonamers.

10. The method of claim 2 wherein said extracting of RNA from said cells comprises

obtaining said cells from said sample;

digesting said cells with proteinase K to produce digested material;

contacted the digested material with a guanidinium containing compound to produce a mixture;

contacting the mixture with a silica matrix to permit RNA binding to the matrix; and

eluting the bound RNA after removal of unbound matter.

11. The method of claim 10 wherein said heating is for about 3 to about 8 hours.

12. The method of claim 11 wherein said heating is for about 3 hours.

13. A method of diagnosing a patient comprising

obtaining gene expression data from one or more cells of said patient,

comparing said data to a gene expression profile generated by gene expression data from one or more FFPE samples, and

diagnosing said patient as having the disease defined by said gene expression profile.

15. A computer readable medium containing a gene expression profile represented as a data structure, said medium having a plurality of data fields stored on the medium and comprising

a first data field representing gene expression profile data that is to be analyzed, said first data field being stored in a range of addresses in said computer readable medium;

one or more receiver objects that will receive test sample expression data for analysis with said gene expression profile data, each receiver object being stored in a separate range of addresses in said computer readable medium,

wherein each receiver object comprises a data field adapted for storing input information for correlation or analysis with said first data field.

14. The method of claim 16 wherein said gene expression profile is generated by amplification of polyadenylated mRNA from said FFPE sample(s).

16. The medium of claim 15 wherein said first data field is stored in a range of addresses used by one of said one or more receiver objects.

17. The medium of claim 15 further comprising a prompt field adapted for storing one or more data prompts to elicit entry of test sample expression data.

18. The medium of claim 15 wherein said test sample expression data is obtained from cells of a tissue containing sample from a human patient.

19. The medium of claim 15 wherein said gene expression profile data is derived from amplification of polyadenylated mRNA from one or more FFPE samples.

20. A system for correlating gene expression data from an FFPE sample with a disease or treatment outcome experienced by the subject from which said sample was obtained comprising

means for generating said gene expression data by amplification of polyadenylated mRNA from said FFPE sample;

means for identifying one or more gene expression levels correlated with at least one disease or treatment outcome experienced by said subject.