Using FFPE RNA-Seq with 12 marker genes to evaluate genotoxic and non-genotoxic rat hepatocarcinogens.

INTRODUCTION

Various challenges have been overcome with regard to applying 'omics technologies for chemical risk assessments. Previously we published results detailing targeted mRNA sequencing (RNA-Seq) on a next generation sequencer using intact RNA derived from freshly frozen rat liver tissues. We successfully discriminated genotoxic hepatocarcinogens (GTHCs) from non-genotoxic hepatocarcinogens (NGTHCs) using 11 selected marker genes. Based on this, we next attempted to use formalin-fixed paraffin-embedded (FFPE) pathology specimens for RNA-Seq analyses.

FINDINGS

In this study we performed FFPE RNA-Seq to compare a typical GTHC, 2-acetylaminofluorene (AAF) to genotoxicity equivocal -cresidine (CRE). CRE is used as a synthetic chemical intermediate, and this compound is classified as an IARC 2B carcinogen and is mutagenic in , which is non-genotoxic to rat livers as assessed by single strand DNA damage analysis. RNA-Seq was used to examine liver FFPE samples obtained from groups of five 10-week-old male F344 rats that were fed with chemicals (AAF: 0.025% and CRE: 1% in food) for 4 weeks or from controls that were fed a basal diet. We extracted RNAs from FFPE samples and RNA-Seq was performed on a MiniSeq (Illumina) using the TruSeq custom RNA panel. AAF induced remarkable differences in the expression of eight genes (Aen, , , , , , and ) from that in the control group, while CRE only induced expression changes in , as shown using Tukey's test. Gene expression profiles for nine genes (, , , , , , , , and ) differed.between samples treated with AAF and CRE. Finally, principal component analysis (PCA) of 12 genes (, , , , , , , , , , , and ) using our previous Open TG-GATE data plus FFPE-AAF and FFPE-CRE successfully differentiated FFPE-AAF, as GTHC, from FFPE-CRE, as NGHTC.

CONCLUSION

Our results suggest that FFPE RNA-Seq and PCA are useful for evaluating typical rat GTHCs and NGTHCs.