Application of exosome gene, prostate cancer detector, and detection kit and detection device thereof
A detection kit and prostate cancer technology, applied in the field of genetic engineering, can solve the problems of low positive detection rate, large error, elevated PSA, etc.
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Embodiment 1
[0071] Extraction and Identification of Urine Exosomes
[0072] After massaging the prostate, collect 20 mL of morning urine, store at 4°C, centrifuge at 2000g for 10 minutes to remove residual cells and collect the supernatant, and then centrifuge at 10000g for 10 minutes to remove cell debris.
[0073] Take 10mL of the supernatant to a new centrifuge tube, add exosome extraction reagent 1 (Shenzhen Shifangjie Technology Co., Ltd.) with a volume ratio of 2:1; invert and mix well, let stand at 4°C for 5min, and then centrifuge at 10,000g 2min; take the supernatant and transfer it to a new centrifuge tube, add exosome extraction reagent 2 (Shenzhen Shifangjie Technology Co., Ltd.) with a volume ratio of 1:1; after inverting and mixing, stand at 4°C for 1 hour After centrifugation at 12000g for 30 min, the supernatant was discarded; the precipitate was collected, and 0.5 mL of PBS buffer was added to resuspend the precipitate, and the exosome suspension was prepared by blowing a...
Embodiment 2
[0076] Exosome gene detection
[0077] 2.1 Extraction of urine exosome RNA:
[0078] ①Take 0.5mL of exosome resuspension and put it into a 1.5ml RNA-free centrifuge tube, add 1mL Trizol lysate, vortex and mix, and let stand at room temperature for 5mins;
[0079] ② Add 200 μL chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 2 minutes;
[0080] ③ After centrifugation at 12000×g for 15 minutes at 4°C, divide into three layers, transfer the upper aqueous phase to a new 1.5mL RNA-free EP tube, be careful not to touch the white membrane in the middle layer;
[0081] ④ Add pre-cooled 0.6mL isopropanol to the tube, mix well, and let stand at room temperature for 10mins;
[0082] ⑤Centrifuge at 12000×g for 10mins at 4°C, discard the supernatant, and sink the RNA to the bottom of the tube;
[0083] ⑥ Add 1 mL of pre-cooled 75% ethanol diluted with sterilized DEPC water, suspend the precipitate, shake gently, and wash the RNA;
[0084] ⑦ Centrifuge ...
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