Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples.
Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharide-stimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.
Scicchitano
Marshall S
MS
Department of Safety Assessment, GlaxoSmithKline, 709 Swedeland Road, Mail Stop UE0364, King of Prussia, PA 19406, USA. Marshall.2.Scicchitano@gsk.com
Dalmas
Deidre A
DA
Bertiaux
Melissa A
MA
Anderson
Shawn M
SM
Turner
Leah R
LR
Thomas
Roberta A
RA
Mirable
Rossana
R
Boyce
Rogely W
RW
D001854:Bone Marrow Cells / Q000737:chemistry; D002478:Cells, Cultured; D015925:Cryopreservation; D005404:Fixatives; D005557:Formaldehyde; D020869:Gene Expression Profiling; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding; D011786:Quality Control; D012313:RNA / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000737:chemistry
D005404:Fixatives; D008070:Lipopolysaccharides; D005557:Formaldehyde; D012313:RNA