Related Experiment Video
Updated: Jun 6, 2026
Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material
Published on: August 1, 2018
Analyses and interpretation of whole-genome gene expression from formalin-fixed paraffin-embedded tissue: an
Muhammad G Kibriya1, Farzana Jasmine, Shantanu Roy
1Department of Health Studies, The University of Chicago, 5841 S, Maryland Avenue, MC 2007, Chicago, IL 60637, USA.
Background:
We evaluated (a) the feasibility of whole genome cDNA-mediated Annealing, Selection, extension and Ligation (DASL) assay on formalin-fixed paraffin-embedded (FFPE) tissue and (b) whether similar conclusions can be drawn by examining FFPE samples as proxies for fresh frozen (FF) tissues. We used a whole genome DASL assay (addressing 18,391 genes) on a total of 72 samples from paired breast tumor and surrounding healthy tissues from both FF and FFPE samples.
Results:
Gene detection was very good with comparable success between the FFPE and FF samples. Reproducibility was also high (r2 = 0.98); however, concordance between the two types of samples was low. Only one-third of the differentially expressed genes in tumor tissues (compared to corresponding normal) from FF samples could be detected in FFPE samples and conversely only one-fourth of the differentially expressed genes from FFPE samples could be detected in FF samples. GO-enrichment analysis, gene set enrichment analysis (GSEA) and GO-ANOVA analyses also suggested small overlap between the lead functional groups that were differentially expressed in tumor detectable by examining FFPE and FF samples. In other words, FFPE samples may not be ideal for picking individual target gene(s), but may be used to identify some of the lead functional group(s) of genes that are differentially expressed in tumor. The differentially expressed genes in breast cancer found in our study were biologically meaningful. The "cell cycle" & "cell division" related genes were up-regulated and genes related to "regulation of epithelial cell proliferation" were down-regulated.
Conclusions:
Gene expression experiments using the DASL assay can efficiently handle fragmentation issues in the FFPE tissues. However, formalin fixation seems to change RNA and consequently significantly alters gene expression in a number of genes which may not be uniform between tumor and normal tissues. Therefore, considerable caution needs to be taken when interpreting gene expression data from FFPE tissues, especially in relation to specific genes.
