Method for identifying glycosylated ribonucleic acid glycoRNA based on solid phase enrichment
A ribonucleic acid and glycosylation technology, applied in chemical instruments and methods, biochemical equipment and methods, DNA preparation, etc. Efficiency, the effect of avoiding experimental steps
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Embodiment 1
[0063] A method based on solid-phase enrichment and identification of glycosylated ribonucleic acid glycoRNA, comprising the steps of:
[0064] (1) Extraction and quality inspection of total RNA in cells
[0065] Here, the traditional Trizol method is used to extract total RNA derived from human or animal tissues or cells (collectively referred to as samples). For specific steps, please refer to figure 2 , figure 2 It is a schematic diagram of the extraction of total RNA in the present invention. like figure 2 As shown, the samples were dissolved in chloroform and then extracted by Trizol method. After stratification, isopropanol was added to the aqueous phase (PH<7) to precipitate total RNA, and then total RNA was washed with 70-75% ethanol and precipitated at least twice. Finally, the total RNA precipitate was dissolved with DEPC water to obtain a total RNA solution. The further detailed steps are as follows:
Embodiment 2
[0091] Please refer to figure 1 , figure 1 This is a schematic diagram of the workflow of the preparation of glycoRNA by solid-phase enrichment of glycosylated RNA in the present invention. like figure 1As shown, a solid-phase glycosylated RNA enrichment and N-endonuclease digestion analysis method (SPRNA-N) and O-glycosidase digestion analysis method (SPRNA-O), comprising the following steps:
[0092] (1) Extraction and quality inspection of Total RNA:
[0093] Method 1: Tissue RNA extraction
[0094] Collect biopsies from clinical patients or animals, take 250 mg of tissue, add liquid nitrogen to quickly grind it, transfer it to an RNase-free EP tube, add 1 mL of Trizol reagent (Beyozol, Shanghai), and then add 200 μL of chloroform, shake Shake and mix for 30 s and let stand on ice for 3 min. Put it into a 4°C centrifuge, 12000g, centrifuge for 15min, aspirate 400μL of the supernatant, add an equal volume (400μL) of isopropanol, invert up and down to mix, and let stand ...
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