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Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples
Citation:
Wehmas, L., C. Wood, B. Chorley, C. Yauk, G. Nelson, AND S. Hester. Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 170(2):357-373, (2019). https://doi.org/10.1093/toxsci/kfz113
Impact/Purpose:
The quality of RNA from archival FFPE samples can vary dramatically. Standard methods of assessing RNA quality are inadequate for characterizing FFPE RNA. In this study we evaluated alternative methods for assessing FFPE RNA quality. Our findings indicate that RNA fragmentation analysis and PCR-based methods are more informative than RIN for characterizing FFPE RNA quality for sequencing but also highlight the need for additional criteria or metrics. This work should help advance the use of archival FFPE samples for genomic analyses, enhancing retrospective analysis, reducing the need for de novo mechanistic studies, and facilitating cross-species comparisons.
Description:
Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100–300 nucleotides in size (DV100–300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.
URLs/Downloads:
DOI: Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples