A biomarker syt12 of osteosarcoma and its application
A 1. SYT12, osteosarcoma technology, applied in the field of osteosarcoma biomarkers, can solve the problem of less molecular targets
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Embodiment 1
[0070] Example 1 Screening of Gene Markers Related to Osteosarcoma
[0071] 1. Sample collection
[0072] Six cases of osteosarcoma tissue and paracancerous tissue samples were collected, and the informed consent of the tissue samples was obtained from the patients, and all of them were approved by the organizational ethics committee.
[0073] 2. Preparation of RNA samples
[0074] Take out the tissue samples frozen in liquid nitrogen, put the tissue samples into a pre-cooled mortar for grinding, and extract and isolate RNA according to the instructions in the kit. details as follows:
[0075] 1) Add Trizol and place at room temperature for 5 minutes;
[0076] 2) Add 0.2ml of chloroform, vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5-10min;
[0077] 3) Centrifuge at 12000rpm for 15min, transfer the upper aqueous phase to another new centrifuge tube (be careful not to absorb the protein material between the two aqueous phases), add ...
Embodiment 2
[0089] Example 2 QPCR sequencing to verify the differential expression of the SYT12 gene
[0090] 1. Large-sample QPCR verification of differential expression of SYT12 gene. According to the sample collection method in Example 1, 50 cases of paracancerous tissues and 50 osteosarcoma tissues of osteosarcoma patients were selected.
[0091] 2. RNA extraction The specific steps are as described in Example 1.
[0092] 3. Reverse transcription
[0093] mRNA reverse transcription was performed using the FastQuant cDNA First Strand Synthesis Kit (Product No.: KR106). Specific steps are as follows:
[0094] Prepare a 20μl reaction system under ice bath conditions: 4×FQ-RT Super Mix 5μl, RNA 1μg, RnaseFree ddH 2 Make up to 20 μl with O, incubate at 42°C for 15 minutes, incubate at 95°C for 3 minutes, and place on ice.
[0095] 4. QPCR amplification
[0096] (1) Primer design
[0097] The primers for QPCR amplification were designed according to the sequences of the SYT12 gene an...
Embodiment 3
[0103] Example 3 Differential expression of SYT12 protein detected by protein immunoblotting
[0104] 1. Extraction of total tissue protein
[0105] Cut the tissue into pieces with scissors, put it into a glass homogenizer, add RIPA lysate, crush the tissue with the glass homogenizer until it is fully lysed, suck the lysed liquid into the EP tube, centrifuge at 14000rpm at 4°C for 5min, Collect the supernatant.
[0106] 2. Determination of total protein concentration
[0107] The protein concentration was determined according to the instructions of the BCA protein concentration determination kit.
[0108] 3. SDS-PAGE electrophoresis
[0109] Prepare 8% separating gel and 5% stacking gel according to the instructions of the SDS-PAGE gel preparation kit and perform electrophoresis.
[0110] 4. Western blot detection
[0111] 1) Electrotransfer
[0112] Put the PVDF membrane into methanol solution to activate for 5 minutes, and put it into the transfer buffer to equilibrate...
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