A molecular marker associated with the occurrence and development of gastric cancer
A gastric cancer and material technology, applied in the field of biomedicine, can solve the problems of early detection of gastric cancer, the impact of recurrence rate and metastasis rate, etc.
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Embodiment 1
[0081] Example 1 Screening of gene markers associated with gastric cancer
[0082] 1. Sample collection
[0083] 8 cases of gastric cancer paracancerous tissues and gastric cancer tissue samples were collected, and 3 cases were normal gastric tissues. All cases did not receive chemotherapy and radiotherapy before surgery. All patients had informed consent and obtained the consent of the organizational ethics committee. .
[0084] 2. Preparation of RNA samples
[0085] Tissue RNA was extracted using QIAGEN tissue RNA extraction kit, and the operation was performed according to the specific steps in the manual.
[0086] 3. Total RNA quantification and purity analysis
[0087] Use Bio-Red ultraviolet spectrophotometer to measure the optical density value of total RNA at 280nm and 260nm, when OD 260 / OD 280 When the value of 1.8-2.0, the purity of total RNA is considered reliable and used for the next experiment.
[0088] 4. Microarray analysis of lncRNA expression
[0089]...
Embodiment 2
[0094] Example 2 QPCR sequencing to verify the differential expression of the LINC00941 gene
[0095] 1. Large-scale QPCR verification of the differential expression of the LINC00941 gene. According to the sample collection method in Example 1, 50 cases of gastric cancer paracancerous tissues and 50 cases of gastric cancer tissues and 10 cases of normal gastric tissues were selected.
[0096] 2. RNA extraction
[0097] RNA samples were extracted using the QIAGEN Tissue RNA Extraction Kit. For details, please refer to the instruction manual.
[0098] 3. QPCR
[0099] 1) Reaction system:
[0100] Add 1 μl of RNA template, 1 μl of random primers, add double distilled water to 12 μl, mix well, centrifuge at low speed at 65°C for 5 min, then place on ice to cool. Add 4 μl of 5× reaction buffer, 1 μl of RNase inhibitor (20U / μl), 2 μl of 10mM dNTP mixture, and 1 μl of AMV reverse transcriptase (200U / μl); mix well and perform centrifugation;
[0101] 2) Reverse transcription reac...
Embodiment 3
[0119] Example 3 Expression of LINC00941 in gastric cancer cell lines
[0120] 1. Cell culture
[0121] Human immortalized gastric mucosal epithelial cell line GES-1, human gastric cancer cell lines HGC-27, MGC-803, and AGS (all purchased from Guangzhou Lydell Biotechnology Co., Ltd.) in the presence of 10% fetal bovine serum and 1% P / S The RPMI1640 culture is based on 37 ℃, 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, use 0.25% EDTA-containing trypsin for routine digestion and passaging, and take the cells in the logarithmic growth phase for the experiment.
[0122] 2. RNA extraction and concentration determination
[0123] Total cellular RNA was extracted using QIAGEN's cellular RNA extraction kit, and the specific operation was referred to the instructions.
[0124] 3. The specific steps of qPCR are the same as in Example 2
[0125] 4. Results
[0126] The result is as figure 2 As shown, compared with g...
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