## 4 Materials and Methods
### Formalin-Fixed, Fresh-Frozen and Flash-Frozen Tissue Samples  
Formalin-fixed, paraffin-embedded (FFPE) and Tissue-Tek(r) embedded fresh-frozen (FF) archived tissues (2008-2019) included in this study were: (1) FFPE MIBC (\(n=6\)) and matched FF MIBC samples (\(n=6\)); FFPE MIBC samples (\(n=23\)); (2) FFPE normal control urothel (\(n=5\)) from non-cancer patients. In addition, five control placental tissues (3 rd trimester) were flash-frozen into liquid nitrogen. Tissue Processing and Methylene Blue (MB) Staining of Tissues for Histology, Micro-Dissection and RNA Isolation  
_Tissue de-paraffinization and re-hydration:_ All tissues were processed according to our standard methodology: FFPE 10 \(\upmu\)m tissue cuts were first treated at 70 \({}^{\circ}\)C for 1 h in a dry oven to facilitate paraffin melting. Tissue cuts were then incubated for 10 min at RT in a sequential order with the following solutions for further de-paraffinization and rehydration: (1) 3 \(\times\) Roti-Histol ([PERSON], #6640.4); (2) 100% Ethanol ([PERSON], #5054.3); (3) 96% Ethanol; (4) 70% Ethanol and (5) dH\({}_{2}\)O with 0.1% DEPC. Matched FF 10 \(\upmu\)m tumor tissues were incubated 2 times for 5 min with 70% Ethanol and dH\({}_{2}\)O to remove Tissue-Tek(r). _MB tissue staining:_ Following tissue de-paraffinization and re-hydration, tissue cuts were incubated with or without MB (1% solution, [PERSON] #AE64.1) for: (1) FFPE and FF 10 \(\upmu\)m tissue sections on glass slides to demonstrate specific histological staining and tumor quantity (Figure 1); or (2) RNA isolation from microdissected MB stained tissue regions. For staining, tissues were incubated for 20 s in MB 1:3 diluted in DEPC treated tap-H\({}_{2}\)O (the time was empirically determined in order not to saturate the tissue) and washed 2 times each 10 s with sterile dH\({}_{2}\)O (total time \(\sim\)1 min). Then samples were centrifuged (16,000\(\times\)_g_ for 5 min at RT) to remove the dH\({}_{2}\)O, and the first solution of the corresponding RNA method was added. _Hematoxylin/Eosin (H&E) tissue staining:_ A modified shortened protocol for H&E histological staining of tissue cuts was used: 1 mL hematoxylin ([PERSON], Taufkirchen, Germany #GHS332) was added to the slide and incubated for 3 min at RT. The slides were washed 2 times with dH\({}_{2}\)O (each 1 min) and 1 mL of bluing buffer (Dako #CS702) for 1 min was added. After 1 time washing with dH\({}_{2}\)O (1 min), 1 mL eosin was added for 1 min at RT, again washed with dH\({}_{2}\)O (total time \(\sim\)10 min). Lastly, all H&E and MB stained tissue slides for histological analyses were cover slipped with glass slides and a mounting buffer (Eukitt, Orsa-tec, #6.00.01.0001.06.01.01). RNA Isolation from FFPE and Tissue-Tek(r) FF Matched Tumor Tissue Samples Using Different Methodologies  
Six MIBC FFPE and matched FF tumor tissues (\(n=6\)), each 10 \(\upmu\)m thick, were stained either with or without MB as above and then immediately processed using different RNA isolation methods to determine RNA integrity. Regarding all methodologies below, the Proteinase K digestion step was extended to overnight for all FF tissues, if applicable. The four RNA isolation methods are shown in Table 1 and explained below with some modifications. The following total RNA isolation methodologies were used for comparison: **(1)** Maxwell RSC DNA Blood kit (Promega, #AS1400) (Method A). For RNA tissue extraction a purchased incubation buffer (Promega, #D920B) was added replacing the DNA extraction buffer. Briefly, samples were heated to 80 \({}^{\circ}\)C for 10 min, then Proteinase K and Protector RNase Inhibitor (Roche, #3335399001) were added, followed by incubation at 56 \({}^{\circ}\)Cfor 1 h while shaking. Afterwards, an RNA lysis buffer (Promega, #Z3051) was added and the samples were pipetted into the cartridges (supplied by the manufacturer) and processed by a Maxwell RSC machine.