Application of gene SUCLG1 or expression products thereof and detection kit
A technology for detection kits and expression products, applied in the biological field, can solve the problems of unclear specific mechanism of renal cell carcinoma
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Embodiment 1
[0032] Example 1 Expression of SUCLG1 in Different Renal Cancer Cell Lines
[0033] Place the cell culture dish on ice, remove the medium, and wash the cells three times with PBS. use Kit (Fast gene, Inc.; Cat. No. 220010) was used to extract total RNA from OSRC-2, A498, 786-D, Caki-1, ACHN, and 769-P cells, respectively. Following the manufacturer's instructions, use a DNA eraser with The RT kit (Takara BioInc.) reverse-transcribed the extracted RNA into cDNA, respectively using cDNA as a template and using SEQ ID NO: 1 and SEQ ID NO: 2 as primers, and performing real-time fluorescent quantitative PCR according to conventional methods and steps. see results figure 1 .
[0034] forward 5'-GAGCAACGGCTTCTGTCATTT-3' (SEQ ID NO: 1);
[0035] reverse 5'-TGCTTGACTCGTACCATGTCC-3' (SEQ ID NO: 2).
[0036] According to the above steps, the total RNA of kidney cancer tissue was extracted, the reverse transcribed cDNA was used as a template, and SEQ ID NO: 1 and SEQ ID NO: 2 were...
Embodiment 2
[0039] Example 2 Effect of overexpression of SUCLG1 on the proliferation of renal cancer cells
[0040] In order to measure cell viability, the plasmid pSUCLG1 overexpressing SUCLG1 was constructed to transfect OSRC-2 and A498 cell lines, respectively, and the transfected cells were cultured and counted in a 96-well plate (1000 cells per well). The time for cell culture and counting is 0, 24, 48, 72, and 96 hours, and the cell viability is measured using the cell counting kit CCK8 analysis kit, and the operation is performed according to the steps of the kit instructions. see results Figure 4 .
[0041] Use the edU cell proliferation detection kit to detect the cell proliferation of the cells cultured for 24 hours, and perform the test according to the steps in the instructions, and the results are shown in Figure 5 , where, vector is the control group transfected with empty plasmid.
[0042] Depend on Figure 4~5 The results showed that the overexpression of SUCLG1 gene...
Embodiment 3
[0043] Example 3 Effect of Overexpression of SUCLG1 on Migration and Invasion of Renal Cancer Cells
[0044] (1) transwell experiment
[0045] Construct OSRC-2 and A498 cells overexpressing SUCLG1, digest with trypsin, centrifuge at 900×g for 3 minutes at room temperature, discard the supernatant, resuspend the sample in RPMI-1640 medium, and incubate at 900×g Centrifuge for 3 minutes and wash cells with PBS. The cells were resuspended in 500 μl RPMI-1640 medium, and the cell density was measured with a hemocytometer. In the invasion assay, the Transwell membrane was wrapped with Matrigel. A total of 2×10 4 cells were placed in the upper chamber of the 8 μm microwell Transwell insert, 500 μl of RPMI-1640 supplemented with 10% FBS was added in the lower chamber, and then the cells were cultured. Migration and invasion were determined by counting the number of cells that successfully migrated through the cell membrane (migration) or through the Matrigel matrix (invasion). Af...
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