RNA samples should not contain organic solvents that inhibit enzymes (such as reverse transcriptase) and excessive concentrations of metal ions, avoiding the contamination of other biomacromolecules such as proteins, polysaccharides and lipid molecules, and eliminating the contamination of DNA molecules.
RNA purification method and precipitation
Organic solvent extraction
Extraction
When using RNA extraction reagent for RNA extraction, it is often used for extraction to remove impurities such as sucrose and protein, and to promote the separation of the aqueous phase and the organic phase, thereby achieving the purpose of purifying RNA. After extracting RNA, isopropanol or ethanol is generally used to precipitate aqueous phase RNA. Add 0.6 times aqueous phase volume of isopropanol or an equal volume of isopropanol with water, precipitate at room temperature for 20-30 min, and centrifuge at high speed to obtain RNA precipitation.
Washing sediment
The RNase-free 75% ethanol was added, and the RNA precipitate was shaken and suspended to sufficiently dissolve the salt ions in the RNA precipitation. RNA was then precipitated again after centrifugation for 10-30 min. After centrifugation, carefully pour off the supernatant (be careful not to pour out the RNA precipitate), then briefly centrifuge, collect the ethanol remaining on the tube wall to the bottom of the tube, suck it with a small pipette, and dry it in the clean bench for 1-2 minutes. Be careful not to dry too much, otherwise the RNA precipitate will not dissolve easily).
Dissolved precipitate
Add an appropriate amount of RNase-Free Water to dissolve the RNA pellet.
Silicon substrate adsorption method
With the improvement of experimental methods, a method for purifying nucleic acids using an adsorbent material has been developed. Currently more common are: silicon matrix adsorbent materials, anion exchange resins and magnetic beads. The silicon matrix adsorption material is the first choice for nucleic acid purification because of its convenient adsorption of nucleic acid, convenient and rapid use, and no use of toxic solvents such as phenol.
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