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 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.