## 5 Conclusions  
In conclusion, we outline several messages when isolating RNA from archival tissues. MB can easily be used for staining tissues of interest for microdissection and RNA isolation resulting in no artifacts due to its chemical properties. We recommend employing MB when extracting RNA from FF tissues especially using Methods B and C, which leads to improved RNA integrity and reference gene expression, like _18 SrRNA_ and _5 SrRNA_ and _5.8 SrRNA_. When isolating RNA from more commonly available FFPE archival tissues, implementing MB becomes a choice depending on the amount of targeted tissue within a sample for RNA isolation and analyses. This becomes important since most methods extracting RNA from FFPE tissues did not show any differences in RNA integrity with or without MB. However, when using microdissection, MB helps to identify specific tissue regions leading to enrichment of an RNA population. As a recommendation, staining with MB and implementing Methods B or C results in excellent _18 SrRNA_ using qPCR, which also demonstrated 100% congruence of MIBC tumor subtyping gene expression using direct multiplex RNA hybridization compared with IHC and mRNA sequencing. Our above recommendations could also be important for spatial sequencing and laser dissection.  
The supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ijms231810267/s1](https://www.mdpi.com/article/10.3390/ijms231810267/s1)]([https://www.mdpi.com/article/10.3390/ijms231810267/s1](https://www.mdpi.com/article/10.3390/ijms231810267/s1)).  
**Author Contributions:** Conceptualization: M.E., P.L.S., R.S. Data curation: M.A., F.F., M.E., R.S. Formal analysis: M.A., F.F., M.E., P.L.S., R.S. Funding acquisition: A.H., R.S., F.F., M.E. Investigation: S.A.K., L.B., M.E., P.L.S., L.G., R.S. Methodology: S.A.K., L.B., M.E., P.L.S., A.B.E., M.A., F.F., R.S. Resources: M.E., V.B., A.H., M.W.B., R.S. Supervision: M.E., P.L.S., R.S. Writing--original draft: P.L.S., R.S. Writing--review and editing: S.A.K., L.B., P.L.S., L.G., A.B.E., M.A., F.F., V.B., A.H., M.W.B., M.E., R.S. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg (Advanced Project D36).  
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the Friedrich-Alexander-University of Erlangen-Nurnberg (252_20 B; 302_19 BC; 353_15 B from 2020).  
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.  
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  
We are grateful for all of the patients who donated their tissues for this study.  
The authors declare no conflict of interest. No funding from commercial sources was obtained for the present study. However, AH had honoraria for lectures for Abbvie, AstraZeneca, Biontech, BMS, Boehringer Ingelheim, Cepheid, Diaceutics, Ipsen, Janssen, Lilly, MSD, Nanostring, Novartis, Roche, 3 DHistotech; advisory role for Abbvie, AstraZeneca, Biontech, BMS, Boehringer Ingelheim, Cepheid, Diaceutics, Gilead, Illumina, Ipsen, Janssen, Lilly, MSD, Nanostring, Novartis, Q Qiagen, QUIP GmbH, Roche, 3 DHistotech; research funding from AstraZeneca, Biontech, Cepheid, Gilead, Illumina, Janssen, Nanostring, Qiagen, QUIP GmbH, Roche, STRATIFYER. ME had personal fees, travel costs and speaker's honoraria from MSD, AstraZeneca, Janssen-Cilag, Cepheid, Roche, Astellas, Diaceutics; research funding from AstraZeneca, Janssen-Cilag, STRATIFYER, Cepheid, Roche, Gilead; advisory roles for Diaceutics, MSD, AstraZeneca, Janssen-Cilag, GenomicHealth. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.