Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, researchers from the U.S. Environmental Protection Agency evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r2 = 0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (<5% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r2 = 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older lower-quality FFPE samples. This work should help advance the use of archival resources in chemical safety and translational science.
Concordance in RNA-seq profiles between 2-year-old FFPE and FROZ samples
A, Principal component analysis (PCA) shows a consistent shift in FFPE compared to FROZ samples. Control FROZ and FFPE samples are located at the upper right (oval). The dotted lines connect FROZ and FFPE sample pairs from the same liver. The asterisk (*) indicates single FROZ outlier sample with high coverage. The PCA plot was generated using RSEM-based output. B, Gene distribution by individual sample shows modest shifts in read alignment to different genomic regions for FFPE samples but general consistency within each sample type. C, Regression plots show high correlation in log2 fold-change (FC) values for common differentially expressed genes between FFPE and FROZ samples, stratified by DEHP dose group.