RNAscope Multiplex FISH Signal Assessment in FFPE and Fresh Frozen Tissues: The Effect of Archival Duration on RNA Expression
Issued Date
2025-01-01
Resource Type
ISSN
00221554
eISSN
15515044
Scopus ID
2-s2.0-85216112376
Journal Title
Journal of Histochemistry and Cytochemistry
Rights Holder(s)
SCOPUS
Bibliographic Citation
Journal of Histochemistry and Cytochemistry (2025)
Suggested Citation
Sari A.I.P., Copeland K., Nuwongsri P., Pipatsakulroj W., Jinawath A., Israsena N., Lertsittichai P., Chirappapha P., Shiao M.S., Jinawath N. RNAscope Multiplex FISH Signal Assessment in FFPE and Fresh Frozen Tissues: The Effect of Archival Duration on RNA Expression. Journal of Histochemistry and Cytochemistry (2025). doi:10.1369/00221554241311971 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/103141
Title
RNAscope Multiplex FISH Signal Assessment in FFPE and Fresh Frozen Tissues: The Effect of Archival Duration on RNA Expression
Corresponding Author(s)
Other Contributor(s)
Abstract
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 (R2 = 0.35 and R2 = 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.