Kit for rectal cancer detection
A kit and rectal cancer technology are applied in the field of rectal cancer detection kits to achieve the effects of high sensitivity, low cost and easy preparation
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Embodiment 1
[0013] Example 1, screening and identification of rectal cancer markers
[0014] 1. Sample collection
[0015] 5mL peripheral blood samples were collected from 15 patients with rectal cancer clinically identified, 13 patients with right colon cancer and 10 patients with left colon cancer, anticoagulated and stored at -80°C. The patients mentioned above were all from the Cancer Hospital of Tianjin Medical University, and signed informed consent with the patients, which also met the ethical requirements.
[0016] 2. RNA extraction
[0017] Total RNA was extracted from colorectal cancer tissues and normal control tissues according to the operation instructions of the Trizol Reagent kit, dissolved in DEPC-treated water, and the concentration and purity of total RNA were measured by Nanodrop-1000 nucleic acid and protein analyzer, and stored at -80°C. Through detection, the RNA concentration is generally between 100ug-250ug / mL, which can be used for subsequent library establishme...
Embodiment 2
[0025] Screening and preparation of embodiment 2 MUT protein nucleic acid aptamer
[0026] Design a random nucleic acid library comprising approximately 20 nucleotides at both ends and 36 nucleotides in the middle as follows:
[0027] 5'-TCAAGCATGAATTGCAGAATGAC(N36)AGCAAGTGAGTAGCCAGTAAGCA-3'; N36 represents 36 random nucleotides.
[0028] (1) Firstly, the original random oligonucleotide library is amplified to increase its copy number to facilitate screening. The original random oligonucleotide library was amplified by PCR with upstream primers and biotin-modified downstream primers, using a PCR kit purchased from Shanghai Sangong Company. The reaction system is as follows:
[0029] Upstream primer 100pmol (5'-TCAAGCATGAATTGCAGAATGAC-3')
[0030] Downstream primer 100pmol (5,-biotin-TGCTTACTGGCTACTCACTTGCT-3,)
[0031] Template DNA 10μg (5'-TCAAGCATGAATTGCAGAATGAC(N36)AGCAAGTGAGTAGCCAGTAAGCA-3')
[0032] Wherein, the downstream primer is a biotin-modified downstream prime...
Embodiment 3
[0038] Example 3 The performance measurement of protein binding suitable gametes
[0039] Based on the property of graphene oxide oxidation to adsorb single-stranded DNA, a method for verifying the affinity of oligonucleotide aptamers was constructed. A fixed concentration of the MUT protein target (1 μM) was incubated with a series of corresponding candidate oligonucleotide aptamers of different concentrations (10, 25, 50, 75, 100, 150, 200 uM) in a total volume of 300 uL, Incubate at 37°C in the dark for 2 hours, and use BB buffer instead of the target as a negative control group. After incubation and binding, GO with the optimal dosage ratio was added to adsorb the aptamer that was not bound to the target. After centrifugation, the fluorescence intensity emitted by the supernatant at 520nm under 490nm excitation was measured with an F-7000 fluorescence photometer. Use dark treatment. Taking the fluorescence intensity of the experimental group relative to the negative cont...
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