In this retrospective study, the two subgroups (disease free or relapsed) Selonsertib chemical structure of patients were equally distributed for sex, age, grade and stage (Table 1). Table 1 Case series   Patients   Recurrent Non recurrent Sex        Male 33 32    Female 3 6 Age, years        <70 19 12    ≥70 17 26 Grade        Low 27 28    High 9 10 Stage        Ta 30 31    T1 6 7 All patients gave written informed consent for biological samples to be used for research purposes. The study protocol was reviewed and approved by the ‘Area Vasta’ Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) Ethics Committee. Macrodissection and DNA isolation Five 5-μm-thick sections were obtained from each

paraffin-embedded block. Macrodissection was Staurosporine performed on hematoxylin-eosin stained sections and only cancer tissue was used for DNA isolation. Genomic DNA was purified using QIAmp DNA FFPE Tissue (Qiagen, Milan), according to the manufacturer’s instructions. DNA was also isolated from a human bladder cancer cell line (HT1376) using Qiamp DNA minikit (Qiagen, Milan, Italy), according to the manufacturer’s

instructions. Methylation specific multiple ligation probe amplification (MS-MPLA) MS-MLPA was performed using at least 50 ng of genomic DNA dissolved in 1XTE JAK pathway Buffer (Promega, Madison, WI, USA). DNA isolated from HT 1376 cell line was used as internal control for MS MLPA analysis (Figure 1). The methylation status of 24 tumor suppressor gene promoters was analyzed using the ME001C1 kit (MRC-Holland, Amsterdam, The Netherlands) (Table 2). Two different probes that recognize two different sites of the promoter region were used for genes RASSF1 and MLH. We excluded CDKN2B gene from the analysis because its probe is sensitive to improper Hha1 next digestion in FFPE samples. In brief, DNA was denatured (10 min at 98°C) and cooled at 25°C, after which the probe mix was added to the samples and hybridization was performed by incubation at 60°C for 16–18 h. The reaction was divided equally in two vials, one for ligation and the other for ligation-digestion reaction for each tumor. We added a mix composed of Ligase-65 buffer, Ligase-65 enzyme

and water to the first vial and a mix of Ligase-65 Buffer, Ligase 65 enzyme, Hha1 enzyme (Promega, UK) and water to the second. The samples were then incubated at 49°C for 30 min. At the end of the ligation and ligation-digestion reactions, samples were amplified by adding a mix of PCR buffer, dNTPs and Taq polymerase. The PCR reaction was performed under the following conditions: 37 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 60 sec. The final incubation was performed at 73°C for 20 min. Figure 1 Electropherogram relating to a) undigested and b) digested HT1376 samples with methylation of APC and RASSF1 genes. Table 2 Summary of gene function and chromosomal localization Gene Function Chromosomal localization TIMP metallopeptidase inhibitor 3 (TIMP3) Invasion and metastasis 22q12.