Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling.
Formalin-fixed paraffin-embedded (FFPE) tissue samples are a potentially valuable resource of expression information for medical research, but are under-utilized due to degradation and modification of the RNA. Using a random primer-based RNA amplification strategy, we have evaluated multiple protocols for the extraction and isolation of RNA from FFPE samples. We found that the RecoverAll RNA isolation procedure with three or four slices (ten-microns in thickness), supplemented with additional DNAse, gave optimal results. RNA integrity as assessed by Agilent Bioanalyzer, and amplification of the 28S ribosomal RNA, were predictive for the number of genes detected on Affymetrix arrays. We obtained expression data for colon and lung tumor and normal FFPE samples and matched frozen samples and found a high correlation between frozen and matched FFPE samples (R(2) between 0.82 and 0.89), while the signature sets in tumor versus normal comparisons were also quite similar. QPCR confirmed all 16 of the differential expression results from the microarrays that we tested. Differentially expressed signature genes from tumor versus matched normal FFPE tissue from colon and lung were identified as cancer-related, with 95 colon tumor and 67 lung tumor genes identified, respectively.
Roberts
Lisa
L
Global Pharmaceutical Product Research Division, Dept R4CD, AP10-2, Abbott Laboratories, IL 60064-6099, USA.
Bowers
Jessica
J
Sensinger
Kelly
K
Lisowski
Andrew
A
Getts
Robert
R
Anderson
Mark G
MG
D003110:Colonic Neoplasms / Q000378:metabolism; D005557:Formaldehyde; D005615:Freezing; D020869:Gene Expression Profiling; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000032:analysis / Q000302:isolation & purification; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods*
D012334:RNA, Neoplasm; D012339:RNA, Ribosomal, 28S; D005557:Formaldehyde; D012313:RNA