Programmable thermal cycler

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).

The sub-MICs (½ MIC) of the enzyme extracts (RAP and granulosain I) were used in the genes' expression and synergism assays.
Total RNA was isolated from planktonic cultures of thirteen strains, before and after treatment with the enzyme extracts, using the TRIZOL reagent according to the manufacturer's instructions (Invitrogen, Buenos Aires, Argentina). The isolated RNA was stored at −20°C. The cDNA was obtained from a reaction mixture including random hexamers and 200 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Invitrogen, Buenos Aires, Argentina) and stored at −20°C.
Transcription levels of pathogenic factors such as omp18, ureA, and flaA were determined by RT-PCR. The 16S rRNA amplicon was used as housekeeping.
The PCR amplification was performed in a programmable thermal cycler (BioRad, California, USA), using the primer pairs shown in Table 1 and the protocols described in Table 2. The identification of RT-PCR products was performed with 1.8% agarose gel electrophoresis. The gels were stained with GelRed Nucleic Acid Gel Stain (Biotium Inc. Hayward, CA, USA), visualized under UV light, and photographed. A 100-bp DNA ladder (PBL, Quilmes, Buenos Aires, Argentina) was included as a molecular mass reference. The semiquantification of the DNA amplicons was performed using software of image processing and analysis in Java (ImageJ, Maryland, USA).