1. Prepare the solution
(1) Extraction buffer (0.02M Tris-acetic acid buffer)
Add 2.42 grams of Tris to 900ml of purified water, adjust the pH to 5.0 with acetic acid, and dilute to 1000ml.
(2) Saturated phenol liquid
Melt phenol at 50°C, add an equal volume of extraction buffer, stir evenly to make a saturated phenol solution, and store in the dark.
(3) Precipitation buffer
3M sodium acetate solution, pH5.0
(4) Dissolving buffer
1M NaCl was dissolved in 0.05M Tris-HCl buffer, pH 9.5.
2. Extraction process
1. Raw material selection and pretreatment
Take 50 kg of calf pancreas (under 1.5 years old) and remove non-pancreatic tissues such as fascia, fat and connective tissue at about 25°C, clean it with purified water, and cut it into small pieces of 3-5 cm.
2. Destruction of tissues and cells
Grind the small piece of pancreas with a clean meat grinder, move the minced calf pancreas into a colloid mill, add an equal amount of extraction buffer, crush and grind at 200 μm for the first time, grind at 50 μm for the second time, and grind under vacuum The liquid should be easier to pass through the 200-mesh stainless steel mesh to obtain a homogenate of about 100kg.
3. Extraction of active ingredients
Add 1 times the volume of saturated phenolic liquid to the upper aqueous phase of the homogenate, add 0.5 times the volume of the lower layer phenolic phase of saturated phenolic liquid, heat to 60 ° C, keep the temperature for 120 minutes, cool rapidly, and centrifuge at 4 ~ 10 ° C (4000g, 30 minutes) , collect the supernatant, discard the precipitate (recovery of phenol).
Add 0.6 times volume of chloroform-isoamyl alcohol (volume ratio 24:1) to the supernatant, shake well for 15 minutes, centrifuge (4000g, 30 minutes) at 4-10°C, collect the supernatant of the upper layer, and discard the precipitate in the middle layer , recover the bottom chloroform layer.
Add 0.3 times the volume of chloroform-isoamyl alcohol (volume ratio 24:1) to the supernatant, shake well for 15 minutes, and centrifuge at 4000g at 4-10°C for 30 minutes), collect the supernatant containing Acido Ribonucleico, and discard the middle The layer was precipitated, and the bottom chloroform layer was recovered.
Add 1 volume of precipitation-promoting buffer to the supernatant, then add 2 volumes of ethanol (95%), shake well, place at 4°C overnight, centrifuge at 4-10°C (4000g, 30 minutes), collect the Acido Ribonucleico precipitate, the upper layer The supernatant liquid is ethanol, reclaims ethanol.
In order to further improve the purity of Acido Ribonucleico and reduce the impurity content, the Acido Ribonucleico precipitate was dissolved with a dissolution buffer, added ethanol to a final concentration of 75% (mass), and centrifuged (4000g, 30 minutes) at 4-10°C.
Repeatedly wash the precipitate twice with 75% ethanol, centrifuge at 4-10°C (4000g, 30 minutes), collect the ribonucleic acid precipitate, and place it in 50°C water until there is no alcohol smell (about 1 hour).
The extraction process of the above process can be completed within 16-18 hours, and the extraction rate of Acido Ribonucleico is 3.8g/kg pancreas, which greatly shortens the extraction time and increases the yield by 1-2 times compared with the existing Acido Ribonucleico extraction process. In the process of RNA extraction, there will always be a small amount of DNA impurities. Since DNA molecules are generally relatively large, it will indirectly cause allergic reactions when injected. Due to the improvement of ribonucleic acid extraction efficiency in the present invention, the RNA content in the Acido Ribonucleico product is about 99.7%, and the purity OD260/OD280>1.9, compared with the product extracted by the prior art (the RNA content is 95-96%), the product purity With great improvement, the content of deoxyribonucleic acid “contamination” is reduced, which can improve product safety; moreover, the batch yield in the extraction process is increased by about 30%, the batch scrap rate is reduced from 30% to zero, and the cost is reduced by about 70%.
