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Rectal Cancer Case Study

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Sample collection This work included 93 patients diagnosed with rectal tumor. Fifty patients with non-metastatic cancer rectum (stage I, II, III) and 43 benign tumor patients were collected following approval from the Ethics Committees of Faculty of Medicine, Zagazig University. We obtained informed consent from all patients. Blood samples were collected prior to the beginning of treatment. Pretreatment rectal tumor biopsy specimens were divided into two parts. One part was sent for routine pathology examination, while the other part was frozen at -80° C until detecting of the methylated status in the promoter of MGMT and ERCC1 genes and their gene expression levels. The malignant cases were referred to Clinical Oncology and Nuclear…show more content…
Clinicopathologic characteristics of rectal cancer patients are listed in Table 1.
DNA extraction
DNA was extracted from blood and from tissue by DNA isolation kits (QIAamp DNA Minikit, QIAGEN GmbH, Hilden, Germany) according to the instructions of manufacturer.
Bisulfite modification
DNA was treated with sodium bisulfite with a commercial kit (EpiTect Bisulfite, QIAGEN) following the manufacturer's protocol. Treatment of genomic DNA with bisulfite results in conversion of all unmethylated cytosine to uracil, while leaving methylated cytosine unaffected.

Methylation-specific PCR (MSP) analysis
We performed methylation analysis of the MGMT and ERCC1 promoters using methylation specific primer pair covering the transcriptional start site in the 5' CpG islands according to methods described by Uno et al. and Liu et al. [19, 20] (Table 2).
PCR was done in a final volume of 25 µL containing 5 µL of bisulfate modified DNA, 1 µL of each primer (1 µM), 12.5 µL of 2X Super Hot PCR Master Mix (Bioron, Germany) and 5.5 µL of H2O. PCR protocol was performed at 95 °C for 10 minutes, then 40 cycles at 95◦C for 30 seconds, annealing temperature for 30 seconds at 61°C for methylation and nonmethylation specific primers of MGMT and at 58°C for methylation-specific primers of ERCC1 or at 54°C for nonmethylation-specific primers of ERCC1, 72◦C for 30 seconds, and final
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