## 2 Results
### MB Tissue Treatment Identifies Histological Regions for RNA Microdissection  
MIBC tissues were stained with MB and then specific targeted tumor regions, microdissected for RNA isolation. MB staining was similar to the state of the art hematoxylin and eosin (H&E) regarding the histological appearance (Figure 1). General Characteristics of Different RNA Isolation Methods for FFPE and FF Tissues Treated with or without MB  
For comparison, five different isolation kits were used to extract total RNA from FFPE and FF tissues stained with or without MB (Table 1). Each methodology is described in detail in the Materials and Methods. According to the manufacturer protocols, 5-10 \(\upmu\)m tissue sections can be extracted for RNA using methodologies based upon magnetic beads (Methods A, B, D), column chromatography (Method C) or via an organic solvent (Method E). The cellulose coated paramagnetic beads in Method A are described as having a higher binding capacity and provide an isolation with a cleaner eluate than traditional DNA purification. For Method B, silica coated magnetic beads are used to bind nucleic acids. Germanium coated beads used in Method D guarantee an efficient binding of nucleic acids and a higher yield compared to other suppliers. During column chromatography with Method C, RNA together with chaotropic salts binds to the surface of glass fibers in a filter tube. After DNase I treatment and washing steps, the RNA is eluted by a low salt buffer. Lastly, isolation containing an organic solvent as in Method E facilitates the separation of RNA, DNA and proteins. Additionally, differences exist between the methods regarding handling of the samples. For example, for FFPE tissues proteinase K treatment was added prior to addition of a lysis buffer (Method A) or directly to the lysis buffer (Methods B-D) with a 40-60 min incubation (Methods A, B and D) or overnight (Method C). All FF tissues were incubated overnight with proteinase K regardless of the method. Furthermore, all samples were heat treated at 80 \({}^{\circ}\)C either before (Methods A and D) or after proteinase K treatment (Methods B and C). Methods A and E did not contain a DNAse I treatment, and thus it was added after the initial procedure. One final difference is the automated purification of RNA for Methods A and B. Within an instrumental run time of 40 min the RNA is isolated, eluted, and purified. This requires purchasing a specific instrument resulting in higher costs per reaction compared to the column based Method C, but might reduce human errors and enable large-scale projects. Methods A and B have a total hands-on time of 3 to 3.5 h, respectively, but only 2.5 h for Method C (Table 1). Figure 1: Example of consecutive FFPE tissue sections showing state of the art hematoxylin and eosin (H&E) and similar methylene blue (MB) staining of normal urothel and muscle invasive bladder cancer (MIBC). Top microscopy photos represent normal urothel H&E staining (left) and MB (right). Bottom photos represent MIBC H&E (left) and MB tissue stains (right). Different histological tissue components: surface epithelia cells: umc = umbrella cells; bc = basal cells; Lamina propria; dTS = desmoplastic tumor stroma; IC = invasive carcinoma. Lower right bar = 100 \(\upmu\)m.  
MB Tissue Treatment Shows Advantages during RNA Isolation of MIBC Tissues Compared to Other Dyes with Different Methodologies  
An initial comparison of extracted RNA from two MIBC FFPE tissues using Method A in the presence of MB revealed statistically significant improved _18 SrRNA_ values compared to no MB treatment using qPCR (Figure 2A). The MB molecular structure is an organic chloride salt with 3,7-bis(dimethylamino)phenothiazzin-5-tum as the counterion (C\({}_{16}\)H\({}_{18}\)ClN\({}_{3}\)S). With this in mind, we asked the question, if other dyes with similar molecular structures or nucleic acid binding properties like MB could also significantly affect _18 SrRNA_ changes using the same tissues.