Related Experiment Video
Updated: May 13, 2026
Laser Capture Microdissection of Paraformaldehyde-Fixed Mouse Liver Tissue for RNA Analysis
Published on: April 17, 2026
Preparation of archival formalin-fixed paraffin-embedded mouse liver samples for use with the Agilent gene expression
A Francina Jackson1, Andrew Williams2, Ivy Moffat2
1Mechanistic Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada; Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
Introduction:
Tissue samples are routinely formalin-fixed and paraffin-embedded (FFPE) for long term preservation. Gene expression analysis of archival FFPE tissues may advance knowledge of the molecular perturbations contributing to disease. However, formalin causes extensive degradation of RNA.
Methods:
We compared RNA quality/yield from FFPE samples using six commercial FFPE RNA extraction kits. In addition we compared four DNA microarray protocols for the Agilent 8×60K platform using 16year old FFPE mouse liver samples treated with phenobarbital or vehicle.
Results:
Despite low quality RNA, archival phenobarbital samples exhibited strong induction of the positive control genes Cyp2b9 and Cyp2b10 by quantitative real-time PCR (qPCR). We tested one- and two-color microarray designs and evaluated the effects of increasing the amount of hybridized cDNA. Canonical gene responders to phenobarbital were measurably induced under each experimental condition. Increasing the amount of labeled cDNA did not improve the overall signal intensity. One-color experiments yielded larger fold changes than two-color and the number of differentially expressed genes varied between protocols. Gene expression changes were validated by qPCR and literature searches. Individual protocols exhibited high rates of false positives; however, pathway analysis revealed that nine of the top ten canonical pathways were consistent across experiments. Genes that were differentially expressed in more than one experiment were more likely to be validated. Thus, we recommend that experiments on FFPE samples be done in duplicate to reduce false positives.
Discussion:
In this analysis of archival FFPE samples we were able to identify pathways that are consistent with phenobarbital's mechanism of action. Therefore, we conclude that FFPE samples can be used for meaningful microarray gene expression analyses.
Insights
Gene expression analysis of archival formalin-fixed, paraffin-embedded (FFPE) tissues is feasible. Despite RNA degradation, microarray analysis can identify molecular perturbations and pathways, but duplicate experiments are recommended to reduce false positives.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues are standard for long-term preservation.
- Gene expression analysis of archival FFPE tissues can reveal disease-related molecular changes.
- Formalin fixation significantly degrades RNA, posing challenges for analysis.
Purpose of the Study:
- To evaluate the feasibility of gene expression analysis in archival FFPE samples.
- To compare the performance of commercial RNA extraction kits and microarray protocols for FFPE tissues.
- To determine if meaningful gene expression data can be obtained from degraded RNA in FFPE samples.
Main Methods:
- Six commercial FFPE RNA extraction kits were compared.
- Four DNA microarray protocols for the Agilent 8×60K platform were evaluated.
- 16-year-old FFPE mouse liver samples treated with phenobarbital or vehicle were used.
Main Results:
- Despite low RNA quality, phenobarbital-induced gene expression was detected by qPCR.
- One-color microarray designs yielded larger fold changes than two-color designs.
- Pathway analysis revealed consistent biological pathways across different protocols, and duplicate experiments were recommended to minimize false positives.
Conclusions:
- Meaningful microarray gene expression analyses can be performed on archival FFPE samples.
- Consistent pathways were identified, aligning with phenobarbital's known mechanism of action.
- Careful protocol selection and validation are crucial for reliable results from FFPE samples.
