*4.4. Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) to Assess the Transcription Levels of MAP1B in Cell Lines and UC Samples*

We calculated the fold change in *MAP1B* gene expression of UC tumors relative to that of normal tissues as previously described [27]. We extracted total RNA from cell lines and a pilot batch of cases consisting of 30 UTUCs and 30 UBUCs to quantify the transcription level of *MAP1B* using real-time RT-PCR. Predesigned TaqMan assay reagents (Applied Biosystems, Waltham, MA, USA) were used to assess the mRNA abundance of *MAP1B* (Hs00195485\_m1) using the ABI StepOnePlus™ system (Applied Biosystems, Waltham, MA, USA), for which *POLR2A* (Hs01108291\_m1) was used as the internal control for normalization.
