Gene expression profiling from FFPE samples

• US 7,364,846 B2
• Filed: 12/23/2002
• Issued: 04/29/2008
• Est. Priority Date: 10/11/2002
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Devices
• Inventors: Erlander, Mark G., Salunga, Ranelle C.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Babic; Christopher M.
• Application Number:
  US10/329,282
• Publication Number:
  US 20040072305A1
• Time in Patent Office: 1,954 Days
• Field of Search: 435/6, 435/91.21, 435/91.2, 435/91.1, 536/23.1, 536/24.33
• US Class Current: 435/6.12
• 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 a formalin fixed and paraffin embedded (FFPE) sample, the method comprising

a) extracting said RNA from said cells of an FFPE sample;

b) heating said RNA at about 70°

C. for 2 to 8 hours; and

c) contacting said extracted RNA with a primer, operably linked to a promoter sequence and comprising an oligo dT sequence, under conditions that result in the synthesis of a first cDNA strand complementary to said extracted RNA, wherein a first cDNA strand is synthesized.

2. The method of claim 1 wherein said heating is for about 3 hours.

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

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

5. The method of claim 3 wherein said random primers are hexamers, heptamers, octamers, or nonamers.

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

obtaining said cells from said sample;

and

digesting said cells with proteinase K to produce digested material containing RNA.

7. The method of claim 6 wherein said heating is for about 3 hours.

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

9. The method of claim 6 further comprising synthesis of the second cDNA strand in the absence of random primers.

10. The method of claim 8 wherein said random primers are hexamers, heptamers, octamers, or nonamers.

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

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

13. The method of claim 11 wherein said random primers are hexamers, heptamers, octamers, or nonamers.

14. The method of claim 2 further comprising synthesis of the second cDNA strand by use of random primers.

15. The method of claim 2 further comprising synthesis of the second cDNA strand in the absence of random primers.

16. The method of claim 14 wherein said random primers are hexamers, heptamers, octamers, or nonamers.

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

18. The method of claim 6 further comprising synthesis of the second cDNA strand in the absence of random primers.

19. The method of claim 17 wherein said random primers are hexamers, heptamers, octamers, or nonamers.

20. 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.

21. The method of claim 6 wherein extracting of RNA from said cells further comprises

contacting 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.

22. The method of claim 1 wherein the RNA is heated at a pH of about 8.0 in a solution comprising Tris-HCl.

23. The method of claim 6 wherein the RNA is heated at a pH of about 8.0 in a solution comprising Tris-HCl.