Circular RNA circ-01477 and application thereof
A technology of circ-01477 and rnacirc-01477, applied in the field of nerve regeneration and repair
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Embodiment 1
[0023] Example 1: Expression and identification of circ-01477
[0024] RNA-seq was performed on the spinal cord tissues of SD (Sprague Dawley) rats at different time points (0d, 1d, 3d, 7d, 14d, 21d and 28d) after spinal cord hemisection injury. According to the sequencing results, select circRNAs that are all composed of exons and have significantly different expressions of loops >500nt. The sequencing results analyzed the screened circRNAs, and further verified the existence of circRNAs by PCR. According to the design principles of circRNAs primers, back primers were designed for these circRNAs, and NCBI was used to design primers for each circRNA. Select different annealing temperatures according to the primer information for PCR amplification, and then select circRNAs with the expected band size through agarose gel electrophoresis, send them to the company for sequencing, and compare the sequencing results with the original sequence to verify whether they span the circula...
Embodiment 2
[0048] Example 2: Functional verification of circRNA
[0049] 1. Culture of primary astrocytes
[0050] The spinal cords of red-skinned mice were isolated one day after birth, and transferred to a petri dish filled with isolation buffer (DMEM). The meninges were peeled off under a stereoscope to separate the spinal cord, and transferred to another Petri dish filled with separation buffer (DMEM). The tissue was washed with separation buffer (DMEM / F12), and the tissue was minced with surgical scissors. Add 2ml of 0.25% trypsin in the digestive solution, digest at 37°C for 15min, and shake several times every 5min. Use digestion termination solution (DMEM / F12+10% FBS) to terminate the digestion, centrifuge at 1000rpm for 5min, and discard the supernatant. Add complete medium (DMEM / F12+10% FBS+1% p.s+1% L-glu) to resuspend the cells. Microscopically observe the agglomeration of nerve cells, pass through a cell sieve, and collect the cell suspension. After counting, they were pl...
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