Method for rapidly identifying circRNA
An identification method and RNA primer technology, which is applied in the field of rapid identification of circular RNA, can solve the problems of expensive, high cost, and time-consuming operation of RNaseR, and achieve the effects of simplifying experimental operations, reducing experimental costs, and achieving accuracy
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Embodiment 1
[0025] Such as figure 1 and figure 2 As shown, this embodiment involves using the method of the present invention to identify the expression of circ-Zfp644 in liver tissue, and the specific implementation steps are as follows:
[0026] (1) Extract total RNA: use fresh or cryopreserved 5-20mg mouse liver tissue, quickly put the tissue into a nuclease-free homogenate tube, add 60μL RNA lysate per 1mg-4mg tissue according to the ratio , placed in an ice bath, crushed the tissue cells with a tissue homogenizer, centrifuged at the maximum speed for 5 minutes, carefully sucked the supernatant into another centrifuge tube; added 0.5 times the volume of supernatant ethanol, and Pipette 3-4 times to get the mixture; transfer the mixture to a spin column with a collection tube, centrifuge at 12000×g, 4°C for 1 min, discard the filtrate; add 600 μL RNA washing solution, Centrifuge at ×g, 4°C for 45 s, discard the filtrate; add 50 μL of diluted DNase I and incubate for 15 min; add 600 ...
Embodiment 2
[0041] Such as figure 1 and image 3 As shown, this embodiment relates to the use of the method of the present invention to identify the expression of circ-Smad5 in the cultured adherent cells JB6, and the specific implementation steps are as follows:
[0042] (1) Extraction of total RNA: JB6 cells were purchased from ATCC in the United States, and the basic medium was DMEM, which was mixed with 10% fetal bovine serum to form a complete medium. Inoculate JB6 cells into a 6cm-diameter petri dish; after 48 hours of culture, the cells grow to a confluence of 60-90%, discard the medium, and wash once with PBS; add PBS solution, peel off the cells with a cell scraper, and collect them in In a RNase-free centrifuge tube; centrifuge at 500g for 5min to collect the precipitated cells; scale (per 1.5×10 3 -5×10 6 Cells, add 300μL RNA Lysis Solution) Add RNA Lysis Solution, place in ice bath, break cells with tissue homogenizer; centrifuge at maximum speed for 5min, carefully draw su...
Embodiment 3
[0057] Such as figure 1 and Figure 4 As shown, the present embodiment involves using the method of the present invention to identify the expression of circ-Arid1a in heart tissue, and the specific implementation steps are as follows:
[0058] (1) Extraction of total RNA: Use 5-20 mg of fresh or cryopreserved mouse heart tissue, quickly put the tissue into a mortar with liquid nitrogen for grinding until the tissue is completely ground into powder; transfer the powder sample To the EP tube without RNase, when the remaining liquid nitrogen is about to volatilize, add the RNA lysate in proportion (for every 5-20mg tissue, add 300 μL lysate), put it in an ice bath, and break the cells with a tissue homogenizer; Centrifuge at the maximum speed for 5 minutes, carefully draw the supernatant into another centrifuge tube; add 0.5 times the volume of supernatant absolute ethanol, mix with a pipette 3-4 times; transfer the mixture to a centrifuge with a collection tube In the column, ...
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