{"title":"RNAscope Multiplex FISH Signal Assessment in FFPE and Fresh Frozen Tissues: The Effect of Archival Duration on RNA Expression.","authors":"Ariestya Indah Permata Sari, Katherine Copeland, Pattarin Nuwongsri, Wiriya Pipatsakulroj, Artit Jinawath, Nipan Israsena, Panuwat Lertsittichai, Prakasit Chirappapha, Meng-Shin Shiao, Natini Jinawath","doi":"10.1369/00221554241311971","DOIUrl":null,"url":null,"abstract":"<p><p>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 <i>UBC, PPIB, POLR2A</i>, and <i>HPRT1</i> 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, <i>UBC</i> and <i>PPIB</i>, than in low-to-moderate expressors <i>POLR2A</i> and <i>HPRT1</i> (<i>p</i><0.0001). Analysis of RNA expression over time showed that <i>PPIB</i>, which has the highest signal, was the most degraded in both adjusted transcript and H-score quantification methods (<i>R</i><sup>2</sup> = 0.35 and <i>R</i><sup>2</sup> = 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.</p>","PeriodicalId":16079,"journal":{"name":"Journal of Histochemistry & Cytochemistry","volume":" ","pages":"9-28"},"PeriodicalIF":1.9000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11755420/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Histochemistry & Cytochemistry","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1369/00221554241311971","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/23 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
Journal of Histochemistry & Cytochemistry (JHC) has been a pre-eminent cell biology journal for over 50 years. Published monthly, JHC offers primary research articles, timely reviews, editorials, and perspectives on the structure and function of cells, tissues, and organs, as well as mechanisms of development, differentiation, and disease. JHC also publishes new developments in microscopy and imaging, especially where imaging techniques complement current genetic, molecular and biochemical investigations of cell and tissue function. JHC offers generous space for articles and recognizing the value of images that reveal molecular, cellular and tissue organization, offers free color to all authors.