Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

Table 2 Methylation level of the regulatory region of the PHD3 gene in primary cancerous tissue and histopathologically unchanged tissue sample from patients with CRC

	Total no. of patients	< 1% methylation	1–10% methylation	10–100% methylation	p^a
Histopathologically unchanged tissue	90	63	27	0	p < 0.00001
Primary cancerous tissue		37	31	22

The primary cancerous and histopathologically unchanged tissue samples from the same patients were used for genomic DNA isolation, followed by bisulfite conversion of cytosine to uracil. The DNA fragments of the CpG island were then amplified by three pairs of primers complementary to the bisulfite-DNA modified sequence (Additional file 1, Additional file 2). To determine the percentage of methylation, the HRM profiles of the patients’ DNA PCR products were compared to HRM profiles of the prepared standard PCR products (Figure 3). DNA methylation of the PHD3 regulatory region for each patient was calculated as mean of the percentage of methylation in two DNA amplified fragments where the differences between primary cancerous and histopathologically unchanged tissue where observed. ^a Chi square test.

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