## 1 Introduction  
Although the molecular distinction between fractionated RNA and DNA was successful in the beginning of the 1900's [1] by detection of Uracil instead of Thymine, respectively, bulk isolations of ribonucleic acids date back to the 1940s. These early fractionation studies used acid and alcohol extractions to isolate plant viral RNA [2], or acid with NaCl and ethanol for nucleic acid recovery from bovine pancreas [3]. However, at that time it was not known if the isolated RNA was a single component or a mixture. The establishment of more quantitative and qualitative methods for RNA isolation included new substances like 2 M guantidinium chloride [4], alkaline extraction without alcohol [5], phenol-water extractions [6] and sodium chloride precipitations [7]. Especially, the finding that 4 M guanidinium chloride inhibited pancreatic RNAses [8] resulted in high quality RNA isolation. Furthermore, using chromatographic substances revolutionized the isolation of RNA and DNA becoming the foundation for RNA/DNA binding column purifications. For example, the first columns consisted of methylated serum albumin kieselguhr [9]. Even more superior, protamine-coated kieselguhr [10] columns along with high salt elution demonstrated high quality total RNA and DNA from rat livers, bacteriophages, bacteria and yeast but without RNA or DNA specificities. The methylene blue (MB) dye, a phenothiazazine derivate, was synthesized in the middle of the 19 th century and produces a blue color when solubilized in water. Due to its small molecular weight, fast diffusion properties and affinity for nucleic acids, MB was initially used to detect tuberculosis in lung tissues [11] and it is still clinically used today for sentinel node mapping, detection of methemoglobin, septic shock and neuroendocrine tumors like insulinomas [12]. In addition, MB, which is United States Food and Drug Administration (FDA) approved, exhibits antiseptic action and can protect tissues from anti-inflammatory and anti-oxidant effects [12]. Importantly, MB also showed chemotherapeutic properties as an antimalarial drug [12; 13] and against cancers where MB is used for photodynamic therapy [14; 15; 16]. The specific ability of MB to stain tissues instead MB as a pathological dye and substituted May-Grunwald-Giemsa [17]. It was also shown that MB specifically binds ssDNA and intercalates dsDNA [18]. MB binds tRNA [19] as well as cellular RNA, where it was used for staining electrophoresed RNA in polyacrylamide gels [20]. Furthermore, MB binds dsRNA and perturbates the dsRNA conformation [21]. However, it was noted that pre-staining 4S and 5S RNAs with MB did not change the migration behavior compared to unstained RNA [22]. For decades, tumor tissues archived via formalin fixed and paraffin embedded (FFPE), fresh or flash-frozen (FF) methodologies represented the main pillars for clinical, therapeutic, translational and basic research. Both ways of archiving tumor tissues have pros and cons, but for RNA and DNA sequencing FF tissues are superior regarding RNA quality, although a few laboratories did not find major quality differences compared to FFPE tissues [23]. Research is ongoing to improve existing methodologies for isolation of RNA especially from FFPE tissues. We previously observed that when MB was incubated with FFPE tissue sections following deparaffinization and rehydration, tumor and normal urothel regions could easily be identified and microdissected. Therefore, we considered MB as a positive aid for identification and microdissection of specific tissue regions. However, it is not known if MB influences RNA throughout isolation used for gene expression studies. In the present report, we systematically analyzed muscle invasive bladder cancer (MIBC) tissues using five different RNA isolation methods comparing patient matched FFPE and Tissue-Tek(r) FF tissues pre-stained with or without MB. These isolation methods were based upon magnetic beads, columns or organic solvent extraction.