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Mining the archives: a cross-platform analysis of gene expression profiles in archival formalin-fixed paraffin-embedded (FFPE) tissue.
Webster, A., P. Zumbo, J. Fostel, J. Gandara, S. Hester, L. Recio, A. Williams, C. Wood, C. Yauk, AND C. Mason. Mining the archives: a cross-platform analysis of gene expression profiles in archival formalin-fixed paraffin-embedded (FFPE) tissue. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 154(2):202-213, (2015).
1. Please explain the nature of the study that resulted in this paper or presentation. Bio repositories have immense potential value in characterizing molecular ta1rgets of environmental chemicals. However, use of archival resources has been limited to date by inconsistent methods for genomic profiling from formalin-fixed paraffin-embedded (FFPE) samples. RNA-Sequencing (RNA-Seq)offers a novel way to address this problem. The goal of this study was to evaluate current protocols for generating RNA-Seq data in FFPE samples in comparison with paired fresh-frozen samples. This work was performed as part of the Genomics Committee FFPE Sequencing Work Group of the International Life Sciences Institute Health and Environmental Sciences Institute (ILSI HESI). 2. Why was this study done? This study supports goals of the Adverse Outcome Pathway Discovery and Development (AOPDD)Project Cancer AOP Task (CSS 12.01.lf) within the ORD Chemical Safety for Sustainability (CSS) research program. The primary goal of this task is to develop biomarkers, tools, and methods to identify AOPs for cancer. Results of this study will be used to establish a platform for retrospectively identifying chemical gene targets and pathways in archival samples. These data will in turn be used to inform prospective modeling efforts within the AOP framework. This work supports Key Product 1 (AOP Descriptions) by developing methods for AOP key event discovery and characterization (Product 1.2 - Formal AOP Descriptions). 3. What is the impact to the scientific field in general and the Agency in particular? The early key events in a chemical mode of action (MOA) or AOP have a major impact on cancer classification and human relevance evaluation. By opening up archival resources to genomic analysis,RNA-Seq may allow for retrospective target identification and AOP development, reducing the need for additional mechanistic in vivo studies and enabling cross-species comparisons. In this study we showed that an RNA-Seq protocol with ribosomal RNA depletion provides strong correlation in RNA profiles between recently collected FFPE and fresh-frozen samples at both the gene and pathway level. However, we also observed differences in the quality of extracted RNA from FFPE samples based on fixation method, highlighting the importance of sample preparation. Broadly, this information should be useful to scientists in industry and regulatory settings involved in MOA or AOP development and evaluation. These findings should also inform new ways to access different biorepository samples,providing a rich bioinformatics pipeline for environmental science and chemical safety.
Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for genomic research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation of nucleic acids. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues using two DNA microarray protocols and two whole transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other three methods by having the highest correlations of differentially expressed genes (DEGs) and best overlap of pathways between FRO and FFPE groups. We next tested the effect of sample time in formalin (18 hours or 3 weeks) on gene expression profiles. Hierarchical clustering of the datasets indicated that test article treatment, and not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18-hour and 3-week FFPE samples compared to FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of age in FFPE block on genomic profiles. RNA-seq analysis of 8-, 19-, and 26-year-old control blocks using the ribo-depletion protocol resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, UTR, intronic, and ribosomal fractions of the transcriptome. Overall, our results suggest that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.