RNAscope Multiplex FISH Signal Assessment in FFPE and Fresh Frozen Tissues: The Effect of Archival Duration on RNA Expression

Formalin-fixed paraffin-embedded tissue (FFPET), which is the most widely used pathology archive, usually has low-quality DNA and RNA due to extensive nucleic acid crosslinking. RNA fluorescence in situ hybridization (RNA-FISH) has been increasingly utilized in research and clinical settings to diagnose disease pathology. In this study, the effect of RNA degradation over archival time on RNA-FISH signals in FFPET and fresh frozen tissue (FFT) was systematically assessed. RNAscope multiplex fluorescent assay with the four house-keeping-gene (HKG) probes UBC, PPIB, POLR2A, and HPRT1 was performed on 62 archived breast cancer samples (30 FFPETs and 32 FFTs). As expected, the number of RNAscope signals in FFPETs is lower than in FFTs in an archival duration-dependent fashion. The RNA degradation in FFPETs is most pronounced in high-expressor HKGs, UBC and PPIB, than in low-to-moderate expressors POLR2A and HPRT1 ( p<0.0001). Analysis of RNA expression over time showed that PPIB, which has the highest signal, was the most degraded in both adjusted transcript and H-score quantification methods ( R 2 = 0.35 and R 2 = 0.33, respectively). This proves that although the RNAscope probes are designed to detect fragmented RNA, performing a sample quality check using HKGs is strongly recommended to ensure accurate results: