Bladder cancer marker lncRNA TERC based on urine exosomes and application thereof
An exosome and bladder cancer technology, applied in the field of bladder cancer marker lncRNATERC, can solve the problems of bladder cancer markers that have not been reported, and achieve excellent diagnostic performance, non-invasive and easy access, and high patient compliance
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Embodiment 1
[0027] Example 1 Screening and extraction of exosome target molecule lncRNA—TERC
[0028] 1. In the present invention, exosomes are firstly extracted from the urine samples of 4 patients diagnosed with bladder cancer and 3 healthy individuals obtained from Tongji Hospital in Shanghai. The experimental flowchart of extracting urine exosomes by differential ultracentrifugation, see figure 1 .
[0029] The extracted exosomes are strictly identified by transmission electron microscopy (TEM), nanoparticle size tracking analysis (NTA) and western blot technology in accordance with ISEV2018 standards, and only qualified exosome samples can be used for downstream analysis.
[0030] 2. The specific steps for extracting exosomal RNA are as follows:
[0031] (1) Add 500 μl of anhydrous trizol to the exosome-PBS mixture, vortex and mix well, and then let stand at room temperature for 5 minutes to fully lyse;
[0032] (2) Add 100 μl chloroform, vortex and mix well, place at room tempera...
Embodiment 2
[0048] Example 2 Method for diagnosing bladder cancer based on urinary exosome lncRNA TERC
[0049] 1. Urine exosome extraction (PEG precipitation method)
[0050] (1) Use a 15ml sterile centrifuge tube to collect 8ml of the urine sample to be extracted, and store it at 4°C;
[0051] (2) Centrifuge the sample at 3000g for 15min at 4°C, discard the precipitate and collect the supernatant;
[0052] (3) Carefully transfer the supernatant into another clean 15ml centrifuge tube;
[0053] (4) Centrifuge the sample at 10,000 g for 20 min at 4°C, discard the precipitate and collect the supernatant;
[0054] (5) Carefully transfer the supernatant into another clean 15ml centrifuge tube;
[0055] (6) Add 2ml PEG6000 (ratio: 4:1) to 8ml of urine supernatant, close the cap of the centrifuge tube tightly, mix upside down for about 1min to make the liquid fully mixed, and evenly distribute the liquid into five 2mlEP tubes middle;
[0056] (7) Put it in a refrigerator at 4°C overnight ...
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