Method for purifying RNA (ribonucleic acid) in biological sample by selective filtration column
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A biological sample and filter column technology, applied in the field of RNA purification in biological samples, can solve the problems of repeated rinsing of inorganic salts and the like
Pending Publication Date: 2019-08-02
ANHUI SENPENG BIOTECHNOLOGY CO LTD
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For the purification of RNA in the upper aqueous phase, methods such as isopropanol precipitation, silica gel membrane, magnetic beads, an
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Embodiment 1
[0023] The method for purifying RNA in a biological sample by using a selective filter column is as follows:
[0024] Get 30mg of fresh mouse liver, add RNA lysis buffer component-1ml (48% (w / w) water-saturated phenol, 49.9% (w / w) guanidine isothiocyanate, 1% (w / w) sodium acetate , 1% (w / w) mercaptoethanol, 0.1% (w / w) 8-hydroxyquinoline, the pH value is 4.2, fully homogenate, stand at room temperature for 10min, add 0.2ml RNA lysis buffer component two (chloroform ), vortex and mix well, centrifuge at 14000r / min for 10min, transfer the upper aqueous phase to the inner tube 2 of the selective filter column, centrifuge at 10000r / min for 5min, after centrifugation, add 30ul DEPC to the inner tube of the spin column for treatment Water, after vortexing, transfer the DEPC-treated water to an RNase-free centrifuge tube, and the resulting solution is the RNA solution. Use 1.5% agarose gel, 1×TAE electrophoresis buffer, and analyze by electrophoresis under 30 mA current conditions Fo...
Embodiment 2
[0026] A method for purifying RNA in a biological sample by using a selective filter column, specifically as follows:
[0027] Get fresh soybean sprout root tip and stem tip respectively, grind in liquid nitrogen, get about 50 mg of ground powder to a 1.5 ml centrifuge tube, add RNA lysis buffer component-1 ml (48% (w / w) water-saturated phenol , 49.9% (w / w) guanidine isothiocyanate, 1% (w / w) sodium acetate, 1% (w / w) mercaptoethanol, 0.1% (w / w) 8-hydroxyquinoline, pH value 4.2, fully homogenate, let stand at room temperature for 10min, add 0.2ml RNA lysis buffer component 2 (chloroform), vortex and shake to mix, centrifuge at 14000r / min for 10min, transfer the upper aqueous phase to the inner tube of the selective filter column In step 2, centrifuge at 10000r / min for 5min. After centrifugation, add 30ul of DEPC-treated water to the inner tube of the spin column. After vortexing, transfer the DEPC-treated water to an RNase-free centrifuge tube. The resulting solution is the RNA ...
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Abstract
The invention discloses a method for purifying RNA (ribonucleic acid) in a biological sample by a selective filtration column. The method specifically comprises the following steps of centrifuging anddelaminating the biological sample treated by an RNA lysis buffer at high speed, adding an upper water phase into an inner tube of the selective filtration column, and centrifuging the selective filtration column in a centrifuging machine; adding 5 to 100 microliters of water into the inner tube of the selective filtration column, oscillating for 30s, and transferring a water solution in the inner tube of the selective filtration column into a centrifuge tube without RNA enzyme, wherein the solution in the centrifuge tube is the purified RNA solution; enabling the RNA lysis buffer to lyse thecells of the biological sample, centrifuging and delaminating at high speed, removing cell fragments and DNA (deoxyribonucleic acid), enabling the RNA to completely dissociate in the upper water phase, and enabling the RNA to pass through a bottom ultrafiltration membrane of the selective filtration column, wherein the ultrafiltration membrane only has permeability on micromolecular substances and no permeability on RNA of biological macromolecular substance under the high-speed centrifuging condition, so as to realize the purifying of the RNA of the biological macromolecular substance.
Description
technical field [0001] The invention belongs to the technical field of nucleic acid extraction, and relates to a method for purifying RNA in biological samples by using a selective filter column. Background technique [0002] RNA includes many different types such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA, etc., and each different type of RNA plays an important role in cell metabolism. In addition, there are The genetic material of some viruses is RNA. RNA is one of the key research objects in many fields such as molecular biology, genetic engineering, medicine, pharmacy, and clinical testing. RNA extraction is one of the routine technical methods in the laboratory. [0003] Compared with DNA, the stability of RNA is worse than that of DNA, and the environment and cells are rich in RNase, resulting in RNA degradation is a common problem in the process of RNA extraction. How to inhibit RNase is a prerequisite for successful RNA extraction ...
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