Application of lncRNA as a biomarker in the diagnosis and treatment of lung adenocarcinoma
A technology of lung adenocarcinoma and drugs, applied in the field of biomedicine
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Embodiment 1
[0067] Example 1 Screening of gene markers related to lung adenocarcinoma
[0068] 1. Sample collection
[0069] Collect 8 cases of lung adenocarcinoma adjacent tissue and lung adenocarcinoma tissue samples. Lung adenocarcinoma tumor tissue specimens were collected from the main tumor area, located at the junction of the middle and outer 1 / 3 of the tumor mass and normal tissues. Obvious necrosis and calcification in the center of the tumor and normal lung tissue around the tumor were excluded; normal lung tissue samples adjacent to cancer were taken from the tumor There is no obvious change in the area above the edge of 5cm. All the above specimens were obtained with the approval of the organization ethics committee.
[0070] 2. Preparation of RNA samples (using E.Z.N.A. kit for operation)
[0071] Introduce liquid nitrogen into a mortar, take the tissue obtained above and put it into a mortar, cut it into powder in liquid nitrogen and grind it into powder, cut it and put it in liq...
Embodiment 2
[0093] Example 2 QPCR sequencing to verify the differential expression of LOC101930114 gene
[0094] 1. Perform large sample QPCR verification on the differential expression of LOC101930114 gene. According to the sample collection method in Example 1, 50 cases of adjacent lung adenocarcinoma tissue and 50 lung adenocarcinoma tissues were selected.
[0095] 2. The RNA extraction steps are as described in Example 1.
[0096] 3. Reverse transcription
[0097] 1) Reaction system:
[0098] RNA template 1μl, random primer 1μl, double distilled water to 12μl, mix well, centrifuge at low speed, 65°C for 5min, then cool on ice.
[0099] Continue to add the following ingredients to the 12μl reaction solution:
[0100] 5× reaction buffer 4μl, RNase inhibitor (20U / μl) 1μl, 10mM dNTP mixture 2μl, AMV reverse transcriptase (200U / μl) 1μl; mix well and centrifuge;
[0101] 2) Reverse transcription reaction conditions
[0102] 25℃ for 5min, 42℃ for 60min, 70℃ for 5min.
[0103] 3) Polymerase chain reaction ...
Embodiment 3
[0119] Example 3 LOC101930114 overexpression
[0120] 1. Cell culture
[0121] Human lung adenocarcinoma cell line A549, using RPMI1640 medium containing 10% fetal bovine serum and 1% P / S at 37℃, 5% CO 2 , Cultivate in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% trypsin containing EDTA for routine digestion and passage.
[0122] Digest the cells in the culture flask with trypsin and inoculate them in a 6-well plate to ensure that the number of cells is 2-8×10 5 Pcs / well, add cell culture medium. Overnight, observe the cell density the next day, the cell density above 70% can be transfected.
[0123] 2. Construction of gene overexpression vector
[0124] Synthesize specific PCR amplification primers according to the cDNA sequence of LOC101930114 (NR_134509.1, as shown in SEQ ID NO.1), add HindIII and XhoI restriction enzyme sites to the 5'end primer and 3'end primer, respectively . The cDNA extracted from the blood of patients wi...
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