Application of duxap8 in the diagnosis and treatment of hepatocellular carcinoma
A kind of hepatocellular carcinoma, reagent technology, applied in the field of biomedicine
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Embodiment 1
[0086] Example 1 Screening for Gene Markers Related to Hepatocellular Carcinoma
[0087] 1. Sample collection
[0088] The cancer tissues and corresponding paracancerous tissue samples of 50 HBV-infected patients with hepatocellular carcinoma were collected, and 8 cases were randomly selected for high-throughput sequencing. The patients all gave informed consent. agree.
[0089] 2. Preparation of RNA samples
[0090] Tissue RNA was extracted using QIAGEN tissue RNA extraction kit, and the operation was performed according to the specific steps in the manual.
[0091] 3. Quality analysis of RNA samples
[0092] The above extracted RNA was subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA were detected by Nanodrop2000, the integrity of the 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 ≥20...
Embodiment 2
[0103] Example 2 QPCR sequencing to verify the differential expression of DUXAP8 gene
[0104] 1. Using the previously collected cancer tissue samples and para-cancer tissue samples from 50 patients, the differential expression of DUXAP8 gene was verified by large sample QPCR.
[0105] 2. The RNA extraction steps are the same as in Example 1.
[0106] 3. Reverse transcription:
[0107] 1) Mix 1 μg of total RNA template with 2 μl 10× buffer, 2 μl dATP (10 mM), 0.5 μl polyA polymerase, 0.5 μl ribonuclease (RNase) inhibitor and ribonuclease-free water (RNase free water), volume The last is 20 μl, and incubated at 37°C for 1h.
[0108] 2) Add 1 μl of 0.5 μg / μl Oligo(dT) specific RT primer to the reaction tube, and incubate at 70° C. for 5 minutes.
[0109] 3) Immediately incubate on ice for at least 2 minutes to break the secondary structure of RNA and primers.
[0110] 4) Mix the above 20 μl reaction mixture with 4 μl 5× buffer, 1 μl dNTP (10 mM), 0.5 μl M-MLV reverse transcr...
Embodiment 3
[0125] Example 3 Differential expression of DUXAP8 gene in hepatocellular carcinoma cell lines
[0126] 1. Cell culture
[0127] Human hepatocellular carcinoma cell lines HepG2, Huh7 and normal liver cell line HL-7702 were incubated in DMEM containing 10% fetal bovine serum and 1% P / S at 37°C and 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.
[0128] 2. Extraction of RNA
[0129] Total cellular RNA was extracted using QIAGEN's cellular RNA extraction kit, and the specific operation was referred to the instructions.
[0130] 3. The specific steps of reverse transcription are the same as in Example 2
[0131] 4. The specific steps of QPCR amplification inspection are the same as in Example 2
[0132] 5. Results
[0133] The result is as figure 2 As shown, compared with normal liver cell lines, the expression of DUXAP8 gene was up-regulat...
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