## 3 Discussion
Our study brings forth new knowledge that MB tissue staining prior to RNA fractionation results in improved RNA integrity especially with FF tissues as determined by qPCR. Importantly, we found that MB tissue staining is easily done within 20 s and its emission does not influence RNA optical density measurements or qPCR using SYBR-Green. As both MB and toluidine blue share similar molecular structures and significantly improved _18 SrRNA_ values using qPCR, MB led to an overall improvement of RNA integrity primarily with FF tissues, which was also reflected by qPCR of _5 SrRNA_ and _5.8 SrRNA_ (Figures 2 and 3). Except for isolation Method A, all other methodologies led to no significant _18 SrRNA_, _5 SrRNA_ or _5.8 SrRNA_ differences when FFPE tissues were treated with or without MB. Examining similarities and differences of RNA extracted from FF and FFPE patient matched MIBC tissues treated with or without MB helps to explain the molecular basis of how MB may positively influence RNA integrity. Although FF and FFPE fractionated RNA fragments were generally smaller in size, no significant RIN or DV200 differences were found. Using our new approach of using _5 SrRNA_ and _5.8 SrRNA_ as reference genes detecting less than 200 bp, both genes showed improvement using qPCR from FF tissues treated with MB, but not with FFPE tissues. This not only confirms our _18 SrRNA_ results for both FF and FFPE tissues, but also supports increased preservation of RNA fragments throughout isolation from FF tissues treated with MB. We propose that the overall improvement of RNA integrity, when implementing MB with FF tissues, most likely stems from the lack of formalin and paraffin fixation and crosslinking found for FFPE tissues [41; 46; 47; 48]. With the same time period prior to fixation, RNA from patient matched FFPE and FF tissues without MB treatment, shows a negative effect of formalin reducing RNA integrity (FFPE Ct mean = 17.71; FF Ct mean = 12.88 for _18 SrRNA_; \(n\) = 6 tumors; isolation Methods A-D). In addition, use of MB also showed a statistically significant benefit to RNA integrity when comparing individual isolation methods for FF tissues (e.g., Method C: no MB, Ct mean = 13.99 and with MB Ct mean = 10.04 for _18 SrRNA_) (Figures 2 and 3). Since RNA fragmentation is ongoing in tissues prior to fixation and upon formalin treatment when nucleic acids cross link, specifically RNA, this leads to hydrolysis [41; 47; 48]. Therefore, we support the idea that formalin fixed RNA fragments are preserved in structure, but during isolation the RNA is more resistant to MB in stabilizing its structure and thus the RNA becomes more fragile leading to poorer integrity. This could be due to remaining cross linked residues and residual paraffin explaining why no differences in _18 SrRNA_, _5 SrRNA_ and _5.8 SrRNA_ qPCR were observed for most isolation methods using FFPE tissues with or without MB. On the other hand, we propose that MB RNA intercalation in FF tissues stabilizes and preserves RNA structure from further fragmentation during isolation resulting in a benefit for RNA integrity. In addition to using _5 SrRNA_ and _5.8 SrRNA_ as marker genes for the integrity of smaller RNA fragments below 200 bp, expression of _5 SrRNA_ (via RNA polymerase III) and _5.8 SrRNA_ (via RNA polymerase I) can also represent an advanced quality check of the cellular state. For example, _Tetrahymena_ cells showed 1:1 molar ratios of _55 rRNA_: _5.8 SrRNA_ during log growth phase. In contrast, starved _Tetrahymena_ cells synthesized approximately 15% more _5 SrRNA_ over _5.8 SrRNA_, supporting _5 SrRNAs_ as a biomarker for changes in physiological cellular conditions [49]. Examining _5 SrRNA_:5.8 SrRNA_ gene expression ratios for all RNA isolation methods, FF MIBC tissues demonstrated ratios closer to 1.0, which could reflect more stable conditions with less cellular stress following loss of blood supply prior to fixation for these six bladder tumors (Figure 3C).