DNA separated or purified from human peripheral red blood cells as well as preparation method and application of DNA
A technology of red blood cells and human peripheral blood, applied in the field of biomedicine, can solve problems such as inaccurate results
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Embodiment 1
[0153] Use EDTA anticoagulant tubes (BD, Cat. No. 367863) to collect whole blood from the subject, and place the whole blood in a refrigerator at around 5°C. See Table 1 for the storage time and sample number of the whole blood sample. Add 5ml of PBS to 5ml of whole blood sample Dilute the whole blood to obtain a diluted whole blood sample.
[0154] Add red blood cell stabilizer (red blood cell stabilizer includes EDTA 5%, sodium fluoride 5%, sodium hydroxide solution 2.5%, Tris-HCl buffer system 2.5%) to the whole blood sample and mix.
[0155] Add 3ml of polysucrose solution with a density of 1.077±0.001g / mL into the centrifuge tube, take 3ml of diluted whole blood sample and spread it carefully on the polysucrose solution, centrifuge at room temperature with a horizontal rotor of 1000g for 20min. There will be obvious stratification after centrifugation, use a syringe to penetrate the wall of the centrifuge tube, and transfer the red blood cells containing a small amount of...
Embodiment 2
[0163] Use EDTA anticoagulant tubes (BD, product number 367863) to collect whole blood from normal subjects, place the whole blood in a refrigerator at about 5°C, and divide it into two groups, one group is added with red blood cell stabilizer, and the other group is not added. The optimal experimental conditions in Example 1 were adopted, that is, the volume ratio of the blood sample to the erythrocyte stabilizer was 10:1, and left for 24 hours. The specific sample information and results were shown in Table 2. 5ml of PBS was added to the 5ml whole blood sample to dilute the whole blood sample. blood to obtain diluted whole blood samples.
[0164] Add red blood cell stabilizer (red blood cell stabilizer includes EDTA 5%, sodium fluoride 5%, sodium hydroxide solution 2.5%, Tris-HCl buffer system 2.5%) to the whole blood sample and mix.
[0165] Add 3ml of polysucrose solution with a density of 1.077±0.001g / mL into the centrifuge tube, take 3ml of diluted whole blood sample and...
Embodiment 3
[0173] Blood was collected from 1 case of colon cancer, 1 case of liver cancer, and 6 cases of normal people. After the blood collection, the method in Example 1 was used to extract the DNA in the red blood cells, and the Septin9 gene methylation was detected by Boercheng (Beijing) Technology Co., Ltd. The kit (PCR fluorescent probe method, National Machinery Note 20153401481), according to the instructions, carried out sulfite conversion and methylation PCR detection on the extracted DNA. The results showed that: 6 normal people were negative, and 1 intestinal cancer patient The methylation of DNA Septin9 extracted from erythrocytes was positive, and the methylation of DNA Septin9 extracted from erythrocytes of a patient with liver cancer was positive. Therefore, the DNA extracted from erythrocytes by the extraction method of this application can effectively detect cancer.
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