Gene expression profiling from FFPE samples
View Patent ↗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.
1. A method for global amplification and analysis of polyadenylated RNA from cells of a deparaffinized FFPE (formalin fixed paraffin embedded) sample, said method comprising
contacting RNA extracted from cells, of said deparaffinized FFPE sample, with a primer comprising an oligo dT sequence under conditions that result in the synthesis of a plurality of first cDNA strands containing said primer and of a plurality of complementary second cDNA strands;
amplifying said cDNA strands to produce amplified molecules; and
hybridizing said amplified molecules to a microarray, wherein said hybridizing produces signals that reflect expressed polyadenylated RNA in said cells.
2. The method of claim 1 wherein said RNA is extracted from said cells by a method comprising
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.
3. The method of claim 2 further comprising heating said RNA extracted from said cells at about 70° C. before contacting said RNA with said primer.
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 wherein said second cDNA strands are synthesized by use of random primers.
7. The method of claim 1 wherein synthesis of the second cDNA strands occurs 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. A method of comparing gene expression data to a gene expression profile, said method comprising
obtaining gene expression data by the method of claim 1 ,
comparing said data to a gene expression profile generated by gene expression data from one or more FFPE samples from one or more other subjects, and
determining whether said data is the same or similar as said gene expression profile.
11. The method of claim 1 , wherein said cells are microdissected from said FFPE sample prior to RNA extraction.
12. The method of claim 1 , wherein said FFPE sample is of a fine needle aspirate (FNA).
13. The method of claim 1 , wherein said FFPE sample is of a core biopsy.
14. The method of claim 1 , wherein said FFPE sample is of a needle biopsy.
15. The method of claim 10 , wherein said cells are microdissected from said FFPE sample prior to RNA extraction.
16. The method of claim 10 , wherein said FFPE sample is of a fine needle aspirate (FNA).
17. The method of claim 10 , wherein said FFPE sample is of a core biopsy.
18. The method of claim 10 , wherein said FFPE sample is of a needle biopsy.
19. The method of claim 1 , wherein said FFPE sample was stored, as a paraffin embedded sample, at a temperature greater than 4° C.