The Method Foundation Of Detecting Aberrant Methylation Of P16 Gene By Microfluidic Chips In Lung Cancer Patients

Lung cancer is one of the malignant tumors. The death rate and incidence are very high through the whole world. It is because its lower early diagnosis rate and poor prognosis. The initiation of lung cancer is a multistage and multistep course involved in many genes. To increase the survival rates of lung cancer patients, we must solve the problem that the earlier we make the diagnosis is the better. In recent years, with acquaints to the mechanism of lung cancer, it has a bright future to make early diagnosis by using molecular markers. Methylation is one of the mechanisms that tumor suppressor genes inactivate. Aberrant methylation of p16 gene promoter is thought to an early thing in lung cancer development. The classic method of detecting aberrant methylation is methylation specific PCR. But its sensity is limited, the method is time-consuming, it is not automated, so it cannot be widely used. To develop a quick, easy, accurate, sensitive method is necessary. We expect to make the early diagnosis by the new method of detecting aberrant methylation of p16 gene by microfluidic chips in lung cancer patients. The most important is that the sensity and specificity of this study are obviously improved than the traditional method. The establishment of this method not only improves the sensity and specificity, but also simplifies the operating course, saves time and reduces the needed samples. We use double-blind experiment to detect the aberrant methylation of tumor, plasma of lung cancer patients and some other patients to discuss the sensity and specificity of the new method. So we can establish a new and reliable method to make early diagnosis for lung cancer patients.Method: First we extract DNA from tumor and plasma of cancer patients and some other patients. Second we make base transformation, so the methylated C changes to U, but the normal C does not change. Third we use methylation specific PCR amplify DNA, then we compare the sensity and specificity between traditional agarose gel electrophoresis and new microfluidic chips.Results: 1.Agarose gel electrophoresis detection results: 30 positive controls are all positive, 30 negative controls are all negative. 44 plasma and 40 tumors of lung cancer patients are all negative. 20 plasma of other cancer patients and 20 plasma of some other patients are all negative too. 2.Microfluidic chip detection results: 30 positive controls are all positive, 3 of 30 negative controls are positive, the others are negative. 13 of 44 plasma of lung cancer patients are positive, the other 31 are negative. 11 of 40 tumor of lung cancer patients are positive, the other 29 are negative. 4 of 20 plasma of other cancer patients are positive, the other 16 are negative. 2 of 20 plasma of other patients are positive, the other 18 are negative. The sensity of microfluidic chips is 100%(30/30). The specificity is 90%(27/30). There is statistical difference between microfluidic chips and agarose gel electrophoresis methods.Conclusions: Compared with agarose gel electrophoresis, the sensity of microfluidic chips is 27.5% higher, the specificity is 90%. The sensity will be more reliable if we improve the specificity. Then it helps detect the low quantity aberrant methylation of p16 gene. The new method makes it possible to diagnosis earlier for high risk people by detecting p16 gene aberrant methylation in plasmas. Perhaps it has great significance in predicting prognosis and therapy.