Analysis of gene expression can provide important information related to prognostic or predictive factors in clinical work. Analysis of DNA or RNA isolated from formalin fixed and paraffin-embedded tissue is generally accepted to be difficult due to degradation and crosslinking. Factors affecting mRNA quality such as formalin fixation length and autolysis period before fixation/freezing have not been systematically addressed. In this study, we analyzed the effect of formalin fixation length and autolysis period before fixation/freezing on the expression of several reference genes in formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma cells and tissue. We also used a factorial experimental design to further analyze the interaction of both variables. It was found that mRNA levels can be reproducibly quantified, independent of the tested variables. Our findings confirm that clinical tumor specimens removed by routine surgical procedures and combined with real-time RT-PCR can be used for the analysis of gene expression in routine FFPE materials and provide some useful information related to the achieved tissues largely stored in histopathology departments.
© 2011 Wiley-Liss, Inc.