Detection reagent for non-small cell lung cancer
A technology for non-small cell lung cancer and detection reagents, applied in the field of single-stranded non-coding small molecule RNA, which can solve the problems of patients losing treatment opportunities and other issues
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Embodiment 1
[0046] Serum samples from patients with non-small cell lung cancer and healthy controls were used for high-throughput miRNA sequencing and analysis. The experimental steps are as follows.
[0047] 1) Serum samples were mixed and placed in 10mL EP tubes. Tissue samples were frozen in liquid nitrogen and stored at -80°C.
[0048] 2) Randomly select 4 samples from each of the collected non-small cell lung cancer serum samples in stage I, stage II, stage III, and stage IV and 4 serum samples from normal healthy controls. Shanghai Kangcheng Bioengineering Co., Ltd. conducts serum miRNA high pass Quantitative sequencing and analysis were performed to screen out miRNAs differentially expressed in non-small cell lung cancer serum samples.
[0049] The present inventors found that a single-stranded non-coding small molecule RNA Lci-miRNA is highly expressed in serum samples of non-small cell lung cancer.
[0050] figure 1 Indicates the expression results of Lci-miRNA detected by hig...
Embodiment 2
[0055] Real-time fluorescent quantitative PCR detection of Lci-miRNA. The experimental steps are as follows.
[0056] 1) When the sample is a human serum sample, use the adsorption column method and use miRcute Serum / Plasma miRNA Extraction and Separation Kit DP503 from TIANGEN to extract serum total RNA; when the sample is human tumor tissue, For human paracancerous tissue samples or human normal tissue samples, use the TRIzol method and use TAKARA RNAiso Plus9109 to extract total RNA from tissue samples.
[0057] 2) Use Thermo Fisher NanoDrop 2000 to measure the concentration and purity of RNA, and use formaldehyde denaturing gel electrophoresis for quality inspection to ensure the integrity of the extracted RNA.
[0058] 3) Using the extracted total RNA as a template, according to the one-step method, use HaiGene TaqMan miRNA Reverse Transcription Kit D1801 of Harbin Beiguo Haiji Biotechnology Co., Ltd. to perform reverse transcription to synthesize cDNA. The reaction syst...
Embodiment 3
[0071] Detection of Lci-miRNA expression in non-small cell lung cancer cell lines. The cells used in this example are all from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. The experimental steps are as follows.
[0072] 1) Take the cells (normal human bronchial epithelial cells HBE, human non-small cell lung cancer cell lines A549, NCI-H1299, NCI-H1975 and EPLC-32M1) out of the liquid nitrogen and quickly put them into the opened in advance and set the temperature to In a water bath at 37°C, gently shake the cryotube left and right for about 1 minute, observe that the cells in the cryotube are completely thawed, and then sterilize the countertop of the ultra-clean workbench with 75% alcohol and transfer the cells to the super-clean workbench in the ultra-clean workbench. Put 2mL of GIBCO RPMI-1640 (C11875500BT) medium in a 15mL centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant, and add fresh RPMI-1640 comple...
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