Urine exosome RNA extraction and library construction method for NGS platform
An extraction method and exosome technology, which are applied in the medical and biological fields, can solve the problems of low RNA content samples, poor RNA quality, and inability to build a library effectively, and achieve an optimized library building process, low cost, and easy clinical application. Effect
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Embodiment 1
[0065] The optimization steps for urine exosome isolation are as follows:
[0066] 1.1 Sample preparation
[0067] Collect fresh urine, add 20mL of urine to a 50mL centrifuge tube, put the centrifuge tube into a high-speed centrifuge and centrifuge at 12000g for 15min, and filter the supernatant through a 0.22μm filter membrane for later use.
[0068] 1.2 Reagent preparation
[0069] Prepare PEG separation solution with PEG 8000 and 5M NaCl mother liquor, specifically, dissolve 450g of PEG8000 in water, add 300mL of 5M NaCl solution, and dilute to 1L to obtain PEG 8000 separation solution.
[0070] 1.3 Isolation of exosomes
[0071] Add 10 mL of PEG8000 separation solution to each 20 mL of filtered urine sample, place the sample in a 4°C refrigerator and incubate for 0.5 h, then centrifuge at 12,000 g for 20 min at 4°C under the action of a refrigerated high-speed centrifuge, remove the supernatant, and inspect with the naked eye Visible precipitation is exosome precipitati...
Embodiment 2
[0073] In view of the low total amount of isolated exosomes, and the exosomes and the RNA contained in them are unstable at room temperature, and it has been reported in the literature that storage will lead to the release of exosome contents and RNA degradation. Therefore, the extraction of exosome RNA requires a fast and high-yield method. The present invention provides an optimized method for extracting exosome RNA from urine. The specific steps are as follows:
[0074] 1. RNA extraction by Trizol method
[0075] Place each centrifuge tube containing 550 μL of isopropanol or 2 mL of ethanol in an environment of -20 ° C to fully pre-cool, prepare 1.5 mL centrifuge tubes, and fill several 1 mL Trizol reagents, keep away from light, and write down the number.
[0076] Transfer the separated exosome sediment sample to a centrifuge tube containing Trizol reagent, shake it well, let it stand at room temperature for 10 minutes, add 0.2mL bromochloropropane to the centrifuge tube, ...
Embodiment 3
[0092] The micro-initial library construction method of extracted exosome RNA, the specific operation is as follows:
[0093] 1. Using the method in Example 2 to extract urine exosome sample RNA;
[0094] 2. Library construction:
[0095] 2.1 RNA fragmentation treatment
[0096] Take out the components of NR1 (Tris Buffer, Beads Binding Buffer, purchased from Novozyme) from the environment at 2-8°C, and let it stand to balance to room temperature (Note: All reagents must be mixed upside down before use. mix well, do not vortex at high speed or shake vigorously). Add 10 μL RNA sample to 19.5 μL Frag / Prime Buffer (purchased from Novizyme), and use a pipette to gently pipette 10 times to mix thoroughly. The sample was placed in a PCR instrument, and the fragmentation conditions are shown in Table 4.
[0097] Table 4 fragmentation processing conditions
[0098]
[0099] Note: Do not stop during the process from fragmentation to first-strand cDNA synthesis.
[0100] 2.2 Fi...
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