Method and kit for identifying state of liver cancer
A kit, technology for liver cancer, applied in the directions of biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc.
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Embodiment 1
[0078] Example 1: DNA Extraction
[0079] DNA Extraction Reagent consists of Lysis Buffer, Binding Buffer, Washing Buffer and Elution Buffer. Lysis buffer consists of protein denaturants, detergents, pH buffers and nuclease inhibitors. Binding buffers consist of protein denaturants and pH buffers. The cleaning buffer is divided into cleaning buffer A and cleaning buffer B, and cleaning buffer A is composed of protein denaturant, nuclease inhibitor, detergent, pH buffer and ethanol; cleaning buffer B is composed of nuclease inhibitor, pH buffer and ethanol composition. The elution buffer consists of nuclease inhibitors and pH buffers. The protein denaturant is: guanidine hydrochloride; the detergent is: Tween20; the pH buffer is: Tris-HCl; the nuclease inhibitor is: EDTA.
[0080] This embodiment takes the plasma sample of a liver cancer patient as an example to extract plasma DNA. The extraction method comprises the following steps:
[0081] (1) Take 1 mL of plasma, add ...
Embodiment 2
[0093] Example 2: Bisulfite Treatment of DNA
[0094] Bisulfite treatment of DNA is to use bisulfite reagent to treat the extracted DNA sample. The bisulfite reagent is composed of bisulfite buffer and protection buffer; the bisulfite buffer is bisulfite A mixture of sodium and water; the protection buffer is a mixture of hydroquinone, an oxygen free radical scavenger, and water.
[0095] In this embodiment, the DNA extracted in Example 1 is used as the processing object, and the DNA is treated with bisulfite, and the specific steps include:
[0096] (1) Preparation of bisulfite buffer: weigh 1g of sodium bisulfite powder, add water to prepare 3M buffer;
[0097] (2) Preparation of protection buffer: weigh 1g of hydroquinone reagent, add water to prepare 0.5M protection buffer;
[0098] (3) Mix 100 μL DNA solution, 200 μl bisulfite buffer solution and 50 μL protection solution, shake and mix evenly;
[0099] (4) Thermal cycle: 95°C for 5 minutes, 80°C for 60 minutes, 4°C fo...
Embodiment 3
[0110] Embodiment 3: Detection of DNA methylation level by real-time fluorescent PCR
[0111]In this embodiment, real-time fluorescent PCR is used as an example to detect the methylation level of DNA methylation marker genes. The DNA methylation marker genes detected were BMP4, COX2, EMX1, Septin9, SOCS1 and TSPYL5 genes, and the internal reference gene was ACTB. We have designed a variety of primers and probes, which can be identical to, complementary to or hybridize to the sequence shown in SEQ ID NO: 1-6 or its complementary sequence (treated with bisulfite), respectively. The sequence of the primer set is as follows:
[0112] BMP4 Primer Set 1
[0113] Primer 1: SEQ ID NO 7: 5'-GAACGCGTTGTTTACGGGTTGGAGG-3'
[0114] Primer 2: SEQ ID NO 8: 5'-TCCCCATCTCGCCGTCGCTCT-3'
[0115] Blocking primer: SEQ ID NO 9: 5'-TGTGTTGTTTATGGGTTGGAGGTTGTGATT-C3-3'
[0116] Probe: SEQ ID NO 10: 5'-FAM-TTCCTCCTCCCGCGCTTTCCGTCCG-BHQ1-3'
[0117] BMP4 Primer Set 2
[0118] Primer 1: SEQ ID NO...
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