RNAscope Multiplex FISH Signal Assessment in FFPE and Fresh Frozen Tissues: The Effect of Archival Duration on RNA
Ariestya Indah Permata Sari1,2, Katherine Copeland3, Pattarin Nuwongsri4
1Program in Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
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.
Insights
Formalin-fixed paraffin-embedded tissue (FFPET) shows reduced RNA quality over time, impacting RNA fluorescence in situ hybridization (RNA-FISH) signals. Housekeeping gene (HKG) expression analysis is crucial for accurate RNAscope assay results in archival samples.
Area of Science:
- Molecular Biology
- Pathology
- Genomics
Background:
- Formalin-fixed paraffin-embedded tissue (FFPET) is a standard archive but yields degraded DNA and RNA due to crosslinking.
- RNA fluorescence in situ hybridization (RNA-FISH) is vital for disease diagnosis, but its accuracy depends on RNA integrity.
- Assessing RNA degradation in archival samples is critical for reliable diagnostic outcomes.
Purpose of the Study:
- To evaluate the impact of archival time on RNA integrity and RNA-FISH signals in FFPET and fresh frozen tissue (FFT).
- To determine the effect of RNA degradation on different housekeeping genes (HKGs) using the RNAscope assay.
- To establish the necessity of sample quality checks for accurate RNAscope assay interpretation.
Main Methods:
- Analyzed 62 breast cancer samples (30 FFPET, 32 FFT) using the RNAscope multiplex fluorescent assay.
- Utilized probes for four HKGs: UBC, PPIB, POLR2A, and HPRT1.
- Quantified RNAscope signals and assessed RNA expression degradation over archival duration.
Main Results:
- FFPET samples exhibited lower RNAscope signals compared to FFT, correlating with archival time.
- RNA degradation was more pronounced in high-expressor HKGs (UBC, PPIB) than low-to-moderate expressors (POLR2A, HPRT1) (p<0.0001).
- PPIB showed the most significant degradation, impacting both adjusted transcript and H-score quantification (R² = 0.35 and R² = 0.33).
Conclusions:
- RNAscope assay can detect fragmented RNA, but archival duration significantly affects signal intensity in FFPET.
- Housekeeping gene expression levels vary in their susceptibility to degradation.
- Implementing HKG-based quality control is essential for ensuring the accuracy and reliability of RNAscope results in FFPET samples.
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