BioProject

For decades, formalin-fixing and paraffin embedding (FFPE) has been routinely used to preserve tissue samples for histological analysis. More...

For decades, formalin-fixing and paraffin embedding (FFPE) has been routinely used to preserve tissue samples for histological analysis. Global gene expression analysis of these archival tissues has the potential to greatly advance research attempting to link perturbations in molecular pathways to disease outcome. We investigated 16-year-old FFPE mouse liver samples treated with phenobarbital and created a protocol for their analysis using Agilent gene expression arrays. Despite low quality RNA, archival phenobarbital samples exhibited strong induction of the positive control genes CYP2b9 and CYP2b10 by RT-PCR. We hybridized Universal Linkage System–labelled cDNA libraries to 8x60K Agilent gene expression arrays. We compared one- and two-colour microarray experiments and tested the effects of increasing the amount of cDNA loaded. Canonical gene responders to phenobarbital treatment were measurably induced under each experimental condition (however increasing cDNA input also increased the array background signal). Individual genes were validated by RT-PCR and literature searches, and pathway analysis demonstrated that 9/10 top canonical pathways were consistent across experiments. These analyses suggested that future experiments should be done in duplicate to identify and eliminate false positive genes. We conclude that FFPE samples can be used for meaningful and reproducible gene expression and pathway analyses using microarrays. Overall design: Phenobarbital-exposed mice (n=3) versus Control mice (n= 3) Less...