## 3 Discussion
Alternatively, _5 SrRNA_:5.8 SrRNA_ gene expression ratios were higher than 1.0 for matched tumor FFPE tissues with identical pre-fixation waiting times, suggesting a higher stability of _5 SrRNA_ during formalin induced RNA fragmentation. Note that, for example, _5 SrRNA_ is used as a preferential reference RNA for micro RNA (miRNA) identification in body fluids [50]. Another important advantage of MB is the similar staining of tissues compared to H&E (Figure 1). The relative short staining protocol of MB versus H&E not only has a benefit of less time, but probably also a positive effect for RNA quality. Although we did not compare RNA integrity of MB versus H&E stained tissues, it was shown previously that RNA isolated from H&E stained tissues harbors the risk of RNA damages, especially without RNAse inhibitors [51]. The risk of RNA degradation was also found to be dependent on the time of the H&E procedure, because a reduction from 26 min to 5 min for the H&E staining improved the RNA quality [52]. Thus, MB is similar to H&E staining for histology and the advantage of MB per se for RNA quality could indicate MB as a superior stain for laser capture microdissection or spatial sequencing. Several publications described the identification of reference genes for bladder cancer [53; 54; 55]. For example, using 9 candidate genes and 14 tumor and control tissues, [PERSON] et al. found SDH and _TBP_ as most stable genes using qPCR (Table S3) [53]. Concerning _TBP_ as a suitable reference gene, this was also established in this present investigation using direct multiplex RNA hybridization. Interestingly, we also identified _GUSB_ and _TBP_ as suitable references for the MIBC and control bladder. Considering the additional steps of cDNA synthesis and amplifications, several factors can mask the best reference genes as also mentioned earlier, such as primers and normalization [28]; thus one recommendation could be to analyze reference genes of tissues with direct multiplex RNA hybridizations and confirm using qPCR. MIBC is subtyped into clinically relevant luminal and basal subtypes important for patient prognosis, chemotherapy and immunotherapy response [25]. This is of particular importance, since MIBC subtype assessment is likely to become a routine clinical standard in the near future, if current ongoing clinical trials validate the importance of subtypes prospectively in randomized settings [25]. Currently, several protein or RNA methodologies exist to assess luminal basal molecular subtypes, e.g., protein subtyping using IHC [56] or mRNA consensus subtyping [25]. Our study demonstrates that when using MB to enrich tumor regions for RNA isolation, along with gene expression normalization using high quality reference genes, high concordance rates between IHC and different gene expression tumor subtyping approaches is achieved. Previously, we implemented a larger panel of genes for subtyping both luminal and basal MIBC subtypes using RNA hybridization [30]. In this report, using a minimum of 3 genes to identify each tumor subtype with both RNA methodologies demonstrated a high specificity. Therefore, it is reasonable that microdissection of targeted tissue regions stained with MB leads to an enriched tumor RNA population with a greater specificity of single copy genes. Due to the clinical significance of MIBC subtyping for patient prognosis and therapy response, our findings clearly underline the importance of proper handling of FFPE tissues. These include standardized RNA isolation methods resulting in high integrity as well as use of correct reference genes in order to achieve precise subtyping results using IHC subtyping and/or direct mRNA hybridization or NGS mRNA-seq. This is relevant as usually only FFPE tissue samples are available in routine patient care scenarios.