Optimization of ctDNA extraction of APC gene in blood of patients with Colorectal Cancer

Background and Aim: Colorectal Cancer (CRC) is the third leading cause of cancer mortality. The APC gene mutation is known as an important biomarker in the diagnosis of colorectal cancer at its early stages, which can be extracted from the blood as ctDNA(circulating tumor DNA). ctDNA which is the result of necrosis or apoptosis of cancer cells, can be extracted from plasma or serum as blood cellular free DNA and can be used as a non-invasive method in the study of CRC molecular mechanisms.Methods: For maximum use of ctDNA potential, optimal knowledge and standardization are required. For this purpose, ctDNA extraction method from plasma and serum samples of patients with colorectal cancer was optimized and various variables were considered, including centrifuge time and plasma and serum isolation temperature. To assess the quality of extracted ctDNA from serum and plasma, the optical density (OD) at ۲۶۰ nm wavelength was measured by UV spectrophotometer. Ultimately, after the design of the primers, the specificity of extracted ctDNA from ۱۰ blood samples of patients with colorectal cancer was evaluated by PCR technique for APC gene.Results: The results of the UV spectrophotometer showed that the level of ctDNA in the plasma extracted samples was higher than the serum samples (p <۰.۰۰۲۳). Temperature at this stage has no effect on the ctDNA value, and room temperature is the best temperature for the ctDNA extraction from serum and plasma. Also, the study of centrifuge time at different stages showed that by increasing the centrifuge time in ۲۰۰۰g and ۱۱۰۰۰g, the ctDNA value increases in plasma samples. Finally, after performing PCR for extracted ctDNA from plasma of patients with colorectal cancer, the electrophoresis pattern confirmed the presence of ۱۶۹bp band for APC gene sequence.Conclusion: The results of this study showed that ctDNA evaluation as a non-invasive method can be an efficient and fast way to different stages diagnosis of cancer but Due to the low level of ctDNA in the blood, extraction optimization is very important.

Keywords:

Optimization of ctDNA extraction , Cancer , APC