Application of CXCL2 to preparation of tool for diagnosis or treatment of lung adenocarcinoma
A lung adenocarcinoma and tool technology, applied in the field of tumor diagnosis, treatment, and prognosis prediction, can solve problems such as the inability to meet the early diagnosis of lung cancer
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0073] Example 1 Screening of differentially expressed genes
[0074] 1. Materials:
[0075] The surgically resected cancer tissues and paracancerous tissues of 8 patients with primary lung adenocarcinoma were used as experimental samples. All cancer tissues were pathologically confirmed as lung adenocarcinoma. All patients with primary lung adenocarcinoma had not received radiotherapy and chemotherapy before operation, and the clinical data of all cases were complete.
[0076] 2. Obtaining tissue RNA
[0077] Total RNA was extracted from tissue samples, and the concentration and purity of the extracted RNA were detected by Nanodrop2000, the integrity of RNA was detected by agarose gel electrophoresis, and the RIN value was determined by Agilent2100. The total amount of RNA required for a single library construction is 5ug, the concentration is ≥200ng / μL, and the OD260 / 280 is between 1.8 and 2.2.
[0078] 3. Remove rRNA
[0079] A part (>24%) of the long non-coding RNAs i...
Embodiment 2
[0103] Example 2 Large sample validation of differentially expressed genes screened out
[0104] Based on the results of the previous high-throughput transcriptome deep sequencing, we selected the CXCL2 gene for verification according to the size of the P value.
[0105] 1. Sample collection
[0106] According to the method of Example 1, 45 cases of lung adenocarcinoma tissues and corresponding paracancerous tissues were collected.
[0107] 2. Validation at the mRNA level
[0108] 2.1 Extract tissue RNA
[0109] Step is with embodiment 1.
[0110] 2.2 Reverse transcription
[0111] Reverse transcription using Primescript 1 st strand cDNAsynthesis kit kit, the operation steps are as follows:
[0112] (1) Add the following reaction liquid in the microcentrifuge tube, as shown in Table 1:
[0113] Table 1 Reaction liquid
[0114] Reagent dose RNA 2.0μg dNTP 1.0μl Oligo(dT) 2.0μl RNase free dH 2 o
Add to 10.0μl
[0115] (2)...
Embodiment 3
[0139] Example 3 CXCL2 Gene Overexpression
[0140] 1. Plasmid construction
[0141] Amplification primers are designed according to the coding sequence of CXCL2 gene, and the design of primers is well known to those skilled in the art. Amplify the coding sequence of the full-length CXCL2 gene from the cDNA library of adult fetal brain (clontech company, article number: 638831), insert the above cDNA sequence into the eukaryotic cell expression vector pcDNA3.1, and connect the obtained recombinant vector pcDNA3.1 -CXCL2 was used in subsequent experiments.
[0142] 2. Culture and transfection of lung adenocarcinoma cells
[0143] 2.1 Cell culture
[0144] The lung adenocarcinoma cell line A549 was cultured in RPMI1640 medium and 10% fetal bovine serum.
[0145] 2.2 Cell transfection
[0146] (1) The day before transfection, 0.5-2*10 5 Tumor cells were suspended in 500 μl of antibiotic-free medium and seeded into 24-well culture plates.
[0147] (2) On the day of transfec...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More