We hypothesized that any differences in bacterial profile at tumor sites in contrast to non-tumor sites may indicate its involvement in tumor pathogenesis. We used 16S rRNA based two culture-independent methods, denaturing gradient gel electrophoresis and sequencing to elucidate the total oral microbiota in non-tumor and tumor tissues of OSCC patients. This may facilitate to identify the microbial transition in non-tumor and tumor tissues and understand better the association of bacterial

colonization in OSCC. Methods Subject selection and sampling procedure Twenty oral tissue samples, 10 each from non-tumor and tumor sites of 10 patients with squamous cell carcinoma of selleck kinase inhibitor oral tongue and floor of the mouth, median age 59 years (53% male and 47% female) were www.selleckchem.com/products/VX-770.html obtained from Memorial Sloan-Kettering Cancer Center (MSKCC) Tissue Bank, refer Estilo et al. and Singh et al. [41–43] for clinical details. The subjects had a history of smoking and drinking Palbociclib and were not on antibiotics

for a month before sampling. The study was approved by institutional review boards at MSKCC and NYU School of Medicine and written informed consent was obtained from all participants involved in this study. The tissues were collected following guidelines established by Institutional Review Board at MSKCC and tumors were identified according to tumor-node-metastasis classification by American Joint Committee on Cancer/Union International

Cancer Center. For this study, to have a homogenous sample population and to control the effect of confounding factors on bacterial colonization, we used non-tumor tissue from upper aerodigestive tract as a control, resected 5 cm distant from the tumor area or contralateral side of the same OSCC patient and confirmed histologically as normal mucosae [42]. The tissue samples were processed to include all bacteria (on the surface and within the tissue) to detect the total bacterial diversity in oral mucosa. The samples were procured and stored at −80°C till further analysis. DNA very extraction from tissue samples Tissue specimens were pretreated as mentioned earlier by Ji et al. [44]. Briefly, the tissues were suspended in 500 μL of sterile phosphate-buffered saline (PBS), vortexed for 30 seconds and sonicated for 5 and 10 seconds respectively. Proteinase K (2.5 μg/mL) was added for digestion and incubated overnight at 55°C, if required, homogenized with sterile disposable pestle and vortexed. The bacterial genomic DNA was extracted by modified Epicentre protocol (Epicentre Biotechnologies, Madison, WI) and purified with phenol-chloroform extraction [45]. Samples were analyzed qualitatively and quantitatively by NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). All samples were stored at −20°C till further analysis. For PCR assays, the DNA concentration was adjusted to 20 ng/μL.