Diagnostic marker for colorectal malignant polyp and application thereof
A technology for colorectal cancer and colorectal cancer, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc.
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Embodiment 1
[0083] Embodiment 1, experimental method and material
[0084] sample handling
[0085] For polyp tissues that meet the enrollment requirements, 5-10 times the volume of 10% neutral formalin was added within 30 minutes after sampling, fixed at room temperature, and embedding and FFPE samples were completed within 24 hours.
[0086] Sequencing process
[0087] NGS next-generation sequencing is used to detect the miRNA of the sample: the sample RNA is extracted and purified, 3' and 5' adapters are constructed, and the library is sequenced on the machine.
[0088] qRT-PCR
[0089] Take the FFPE sample and cut a number of 10 μm slices, and select 1 to 3 slices according to the size of the tissue for qRT-PCR detection. The process is as follows: figure 1 shown. The program of reverse transcription into cDNA is: 42°C for 60min; 85°C for 5min; the PCR detection program is: enzyme activation, 95°C, 10min; denaturation at 95°C, 15s; extension at 60°C, 60s; denaturation and extensio...
Embodiment 2
[0093] Example 2, discovery set
[0094] After NGS next-generation sequencing was performed on normal mucosal biopsy specimens, early cancer endoscopic biopsy specimens, and colorectal cancer surgical specimens from the same patient, it was found that the changes in the expression levels of some miRNAs were consistent. Select the following miRNAs as candidate genes: has-miR183-5p, has-miR195-3p, has-miR378a-5p, has-miR150-3p, has-miR584-5p, has-miR21-5p, has-miR182-5p, has -miR214-3p, has-miR30a-3p, has-miR129-5p.
[0095] In order to further search for miRNAs closely related to colorectal cancer, qRT-PCR was performed on 187 historical tissue samples, and 7 miRNAs with relatively significant differences between the experimental group and the control group were screened out of 10 candidate miRNAs: hsa -miR-129-5p (vs. carcinoma: p=0.0757, vs. adenoma: p=0.0134), hsa-miR-150-3p (vs. carcinoma: p=0.0137, vs. adenoma: p=0.0008) , hsa-miR-195-3p (vs. carcinoma: p=0.0048, vs. ade...
Embodiment 3
[0100] Embodiment 3, training set
[0101] 3.1 Sample inclusion criteria:
[0102] standard constrain:
[0103] a) Over 18 years old, male or female;
[0104] b) Colonoscopic biopsy of adenomatous polyps with a diameter ≥ 2 cm, and biopsy, with a clear pathological diagnosis; or surgical resection of the sample, with a clear pathological diagnosis;
[0105] exclusion criteria
[0106] a) People who do not meet the inclusion criteria;
[0107] b) People whose sample background information is not clear;
[0108] c) People with a history of cancer.
[0109] 3.2 Grouping criteria:
[0110] According to the pathological diagnosis of the wax block, the samples were classified into canceration, high-grade neoplasia, low-grade neoplasia, and inflammation;
[0111] Determine the grade of all the wax blocks of the lesion according to the highest pathological grade of the lesion;
[0112] Among them, canceration and high-grade neoplasia are the disease group (positive), and low-g...
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