0 test (Epigenomics AG, Berlin, Germany) according to the manufacturer��s instructions. Bisulfite conversion results in the deamination of unmethylated cytosine nucleotides that are eventually converted to uracil nucleotides. Upon PCR amplification, the unmethylated cytosines are replaced with thymine nucleotides better while the methylated cytosines remain as cytosines. The subsequent real-time PCR detected the methylated CpG sequences within the v2 transcript of the Septin 9 gene and the total bisulfite-converted DNA of a region of the beta actin gene (ACTB). A methylated Septin 9-specific fluorescent detection probe, bisulfite-converted unmethylated sequence specific blocker and primers designed in regions lacking CpG dinucleotides were used for PCR reactions.

Each sample was tested in triplicate during PCR analysis with the LightCycler 480 (Roche Diagnostics, Basel, Switzerland) instrument. In all independent runs, Epi proColon Negative Control and Epi proColon Positive Control were used. Validation limits were used for real-time PCR assays according to the manufacturer��s instructions. Accordingly, for Septin 9 PCR the crossing point (CP) for the positive control was less than 40.5 and the negative control had no CP. For ACTB PCR, the positive control was less than 30.3 CP and the negative control was less than 37.1 CP. The PCR validity limits were different for the patient samples. In the positive cases, Septin 9 PCR CP was less than 50 and ACTB PCR CP was less than 33.7, while the Septin 9 negative cases showed no CP in Septin 9 PCR and ACTB PCR CP was less than 33.

7. All of the amplification curves were regularly shaped; otherwise they were excluded as invalid measurements. To be comparable to previous studies using SEPT9 (deVos et al. [16]), we first analyzed the data using a 1/3 rule in which a sample was declared positive if 1 of 3 PCR replicates had a valid curve (1/3 analysis method). Thus, a sample was considered to be positive for Septin 9 if at least one of the three Septin 9 PCRs were positive and a sample was considered to be negative for Septin 9 if all 3 of the Septin 9 PCR replicates were negative. In addition, we also analysed the data using a 2/3 rule, whereby to be called positive 2 of the 3 PCR replicates must have valid curves, following the instructions for use of the manufacturer. (2/3 analysis method).

Application of this rule results in increased specificity Brefeldin_A at a lower sensitivity of detection. Guaiac-based Fecal Occult Test (gFOBT) All fecal samples for gFOBT (Hema Screen, Immunostics. Inc., New Jersey) were taken at least 2 days before bowel preparation by patients. The test was done in the Central Laboratory of Semmelweis University. The detection level of stool blood was 0.6 mg Hg/gm of feces. Carcinoembryonic Antigen (CEA) All blood samples for the CEA test (Cobas, Roche Diagnostics) were taken at least 2 days before bowel preparation.