Genomic DNA and total RNA were extracted from bladder cell lines and Cohort 2 using AllPrep DNA/RNA Kit (Qiagen, Germantown, MD, USA) and cohort 3 using QIAamp DNA FFPE Tissue Kit (Qiagen). Extracted DNA from Cohort 1 was provided by EDRN. PCR primers were designed to amplify the promoter and each exon in PAI1. DNA were used for PCR amplification. PCR reactions were carried out in a total volume of 25 µL containing genomic DNA template, 0.4 µM of each PCR primer, and GoTaq G2 Hot Start Green Master Mix (Promega, Madison, WI, USA), except for PAI1 exon 9. Due to the length of exon 9′s PCR products, AccuStart II GelTrack PCR Super Mix (Quanta BioSciences, Inc., Gaithersburg, MD, USA) was used. Forty cycles of 30 s at 94 °C, 30 s at 60 °C, and 30–120 s at 72 °C (per intended PCR product) were performed in a programmable thermal cycler (Bio-Rad, Hercules, CA, USA). PCR products were checked on a 1.5% agarose gel, followed by PCR purification and bidirectional Sanger sequencing (Psomagen, Rockville, MD, USA). Sequence analysis was performed using Geneious, (Geneious version 8, “http://www.geneious.com (accessed on 5 August, 2015)”, [51 (link)]) and detected variants were confirmed against a RefSeq genomic DNA sequence (PAI1: NG_013213.1). All genetic alterations identified by Geneious were compared to the NCBI SNP database (dbSNP).
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