## 2 Results
### GUSB, TBP and UBC Are the Most Suited Reference Genes for MIBC and Normal Bladder Utrohelium  
In contrast to qPCR, direct multiplex RNA hybridization is an amplification free, highly multiplexed and throughput technique with significant sensitivity and robustness [37]. In order to determine the most suitable reference genes for MIBC and normal urothel, 34 commonly used reference genes from the literature were tested using direct multiplex RNA hybridization and FFPE tissues (Table S2). We chose Method B for RNA isolation, which showed excellent _18 SrRNA_, _5 SrRNA_ and _5.8 SrRNA_ values in the presence of MB prior treatment (Figure 2B). In addition to the 6 FFPE MIBC tissues we expanded our cohort to a total of 28 MIBC and 5 normal urothel isolated RNAs also using Method B and MB  
Figure 3: MIBC qPCR _5 SrRNA_ and _5.8 SrRNA_ values from RNA isolated from FFPE and FF tissues using different isolation methods. (**A**) Each box graph shows _5 SrRNA_ Ct values (\(Y\)-axis) from RNA isolated from six different FFPE MIBC tissues and patient matched FF MIBC tissues with (+) or without (—) MB prior staining using four different isolation methods (A–D) (Table 1); **\(p=0.0022\)**, \(n\) = 6 technical replicates. (**B**) Each box graph shows _5.8 SrRNA_ Ct values (\(Y\)-axis) from RNA isolated from six different FFPE MIBC tissues and patient matched FF MIBC tissues with (+) or without (—) MB prior staining using four different isolation methods for FFPE (A–D) and five isolation methods for FF (Table 1); **\(p=0.0043\); *\(p=0.026\)**, \(n\) = 6 technical replicates. (**C**) Graph shows ratios (\(Y\)-axis) of _5 SrRNA_/_5.8 SrRNA_ Ct values from RNA isolated from six different FFPE MIBC tissues and patient matched FF MIBC tissues with (+) or without (—) MB prior staining using four different isolation methods for FFPE (A–D) and five isolation methods for FF tissues (Table 1). Far right represents control human placenta (_n_ = 5) (flash frozen tissue in liquid nitrogen and isolated with Method E) used for comparison. for microdissection. All raw counts were first normalized to the positive plate control to minimize technical errors between runs. Subsequently, all expression counts of the MIBC and normal urothel were analyzed for each reference gene by calculating the coefficient of variation (COV) or relative variability, which represents the standard deviation of a group of values divided by their mean. The COV was determined in percent with the lowest COV supporting the best constant reference gene expression (Figure 4A,B). Analysis revealed three reference genes: Glucuronidase beta (_GUSB_; HGNC:4696) (COV 61.44%), Ubiquitin C (_UBC_; HGNC:12468) (COV 61.38%) and TATA-box binding protein (_TBP_; HGNC:11588) (COV 70.99%). _GUSB_ is involved in the degradation of glycosaminoglycans [38]. The TATA box-binding protein (_TBP_) is the DNA-binding subunit of RNA polymerase II transcription factor D, essential for expression of most protein-encoding genes [39]. As a member of the ubiquitin family, Ubiquitin C (_UBC_) is a polyubiquitin precursor, and conjugation of ubiquitin is associated with many cell functions, especially with protein degradation [40]. Finally, these genes were also positively confirmed using qPCR, as indicated by slope values of standard curves for _18 SrRNA_\(-\)3.221 (R\({}^{2}\): 0.997), for _TBP_\(-\)3.55 (R\({}^{2}\): 0.990) and for _UBC_\(-\)3.69 (R\({}^{2}\): 0.979) (Figure S2F-H). Interestingly, comparing the standard curves, slopes and R\({}^{2}\) of RNA isolated from two different MIBC FF tissues further indicates that the isolation technique results in similar RNA concentrations and degradations and confirms highly similar amplification efficiencies and linearity. MIBC Subtyping of Luminal and Basal Tumors Comparing Protein IHC, Direct Multiplex RNA Hybridization and mRNA Sequencing  
MIBC can be subtyped into clinically significant luminal and basal subtypes correlating with patient prognosis and response to chemotherapy [25].