Melanoma metastasis-associated marker and application thereof
A melanoma and genetic technology, applied in the field of disease diagnosis, can solve problems such as rising, diagnosis and treatment gaps
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Embodiment 1
[0033] Example 1 Screening for differentially expressed gene markers
[0034] 1. Sample collection
[0035] Primary group: primary lesion of melanoma, 3 cases
[0036] Metastatic group: melanoma metastatic lesions, the metastatic site is the lungs, 3 cases
[0037] 2. Tissue RNA total extraction
[0038] The tissue RNA extraction kit from TIANGEN Company was used to extract the total RNA. For details, please refer to the instruction manual.
[0039] 3. RNA quality detection
[0040] The purity and concentration of RNA were measured by Nanodrop2000 UV spectrophotometer, and the purity was higher when OD260 / OD280 was between 1.8-2.2. Agilent2100 measures the RIN value.
[0041] 4. High-throughput transcriptome sequencing
[0042] The samples were sequenced using Illumina Hiseq x-ten second-generation high-throughput sequencing technology.
[0043] The software cuffquant and cuffdiff were used for mRNA expression quantification and differential expression analysis. Cuffqu...
Embodiment 2
[0046] Example 2 QPCR verification screened differentially expressed gene markers
[0047] Based on the screening results of Example 1, DEGS2 or NKPD1 was selected for QPCR verification.
[0048] 1. Sample collection
[0049] According to the method of Example 1, 36 cases of melanoma primary lesion tissue and 33 cases of melanoma metastatic lesion tissue were collected.
[0050] 2. Extract tissue RNA
[0051] Step with reference to embodiment 1.
[0052] 3. Synthesize cDNA
[0053] First remove the genomic DNA reaction, add 5×gDNA Buffer 2.0μl, Total RNA 1μg, add RNase Free ddH to the test tube 2 O Bring the total volume to 10 μl, heat in a water bath at 42°C for 3 minutes, then add 2.0 μl of 10×Fast RT Buffer, 1.0 μl of RT Enzyme Mix, 2.0 μl of FQ-RT Primer Mix, RNase Free ddH 2 O 5.0 μl, after mixing, add to the above test tube and mix together a total of 20 μl, heat in a water bath at 42°C for 15min, then at 95°C for 3min.
[0054] 4. QPCR reaction
[0055] (1) Prime...
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