## 3 Discussion  
Routine pathological tissue processing to establish biobanks of archival tissues generally involves FFPE, Tissue-Tek(r) or liquid nitrogen. All tissue processing methods share one critical step, the time period after surgery prior to fixation. This waiting time varies between patient surgeries and understandably cannot be standardized. Following tissue oxygen ablation, rapid RNA fragmentation begins and progresses over time as more anoxic as well as hypoxic conditions due to a reduction of pH prevail [41]. Methods involving immediate freezing stabilize RNA and DNA structure; however, it is well known that formalin preserves RNA and DNA structures by introducing nucleotide crosslinking, especially with proteins, but also results in strand breakage into fragments with an average of 100-200 bp  
Figure 4: Identification of stable reference genes for MIBC and control tissues. (**A**) Using direct multiplex RNA-hybridization of MIBC (\(n=28\)) and control bladder (\(n=5\)), the coefficient of variation (COV) in % was calculated. Line shows mean COV of all reference genes and green points represent the most stable expressed reference genes. (**B**) Graph (\(Y\)-axis) demonstrates the COV % of six different reference genes using qPCR: 55_rRNA_ (sharp circle), 5.85_rRNA_ (square), 185_rRNA_ (triangle) and confirmation of the three most stable reference genes, _TBP_ (triangle), _GUSB_ (diamond) _UBC_ (blurred circle) for combined MIBC and control bladders. Although 185_rRNA_ showed a relative high COV of 21.7%, it represented the gene with the highest detection rate (lowest Cts). (**C**) Unsupervised hierarchical cluster analysis of 28 MIBC based on six subtyping genes (basal differentiation: _KRT5_, _KRT14_, _CD44_; luminal differentiation: _KRT20_, _GATA3_, _FOXA1_) assessed by multiplex RNA-hybridization and normalized according to the three optimal housekeeping genes _GUSB_, _TBP_ and _UBC_. Fourteen samples were classified each as basal or luminal. (**D**) Comparison of multiplex RNA-hybridization subtyping, protein IHC subtyping (derived by IHC assessment of KRT5/CK5, KRT14/CK14, CD44, KRT20/CK20, GATA3 and FOXA1) and transcriptome consensus subtyping. Consensus subtyping is displayed in two categories: (1) “simplified” divided into a luminal and a basal category and (2) “detailed” divided into a basal and four luminal categories (luminal, luminal papillary/Lump, luminal unstable/Lump and stroma-rich). Importantly, concordance rates between multiplex RNA-hybridization subtyping and other approaches amounted to 100%; furthermore, all luminal subsets were correctly classified as luminal by multiplex RNA-hybridization subtyping. length [41; 42; 43; 44]. Furthermore, formalin fixed tissues can lead to additional RNA fragmentation following paraffin embedding upon exposure to light. On the other hand, paraffin can lead to high molecular weight RNA aggregates, resulting in low yield and poor quality RNA [45]. Thus, a limiting factor in retrieving RNA from FFPE tissues is complete paraffin removal, rehydration and protein breakdown to help free nucleotide crosslinks [46; 41]. Even more problematic is the additional RNA damage during fractionation that can occur with the use of various isolation methods. Therefore, the task of isolating RNA from a preserved tissue state is not only challenging as regards choice of the correct method, but also further research is needed to improve RNA integrity. Chemical dyes for the histology of pathological tissues are critical for diagnosis, but can also be an aid for microdissection and enrichment of specific tissue regions for isolation of RNA and DNA needed for molecular studies. To date no systematic studies comparing FFPE with FF human tissues treated with or without MB and addressing its influence on RNA integrity had been performed. Thus, we investigated whether MB staining of FFPE and FF tissues has an influence on RNA integrity, using four or five isolation methods, respectively.