CN111172155A - Method for extracting total RNA from fish adipose tissue - Google Patents
Method for extracting total RNA from fish adipose tissue Download PDFInfo
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- CN111172155A CN111172155A CN202010146687.5A CN202010146687A CN111172155A CN 111172155 A CN111172155 A CN 111172155A CN 202010146687 A CN202010146687 A CN 202010146687A CN 111172155 A CN111172155 A CN 111172155A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention discloses a method for extracting total RNA from fish adipose tissues, which comprises the following steps: step 1: preparing a clean operation tool, step 2: primary treatment of adipocytes, step 3: standard treatment before centrifugation, step 4: centrifugation of adipose tissue, step 5: standard treatment before secondary centrifugation, step 6: the invention relates to the technical field of RNA (ribonucleic acid) and provides secondary centrifugal treatment. According to the method for extracting the total RNA from the fish adipose tissues, after the adipose tissues are physically crushed, the adipose tissues are mixed with a reaction solvent through a dropper, so that the decomposition of the adipose cells is effectively accelerated, the problem that the RNA in the adipose cells cannot be completely dissolved in the dissolving liquid because the tissue cells are not treated in place when the RNA is extracted from the animal body tissues in the past is solved, the RNA in the cells can be completely cracked and released, the RNA naturally released in the cells is greatly increased, and the device is simple in structure and easy to maintain.
Description
Technical Field
The invention relates to the technical field of RNA, in particular to a method for extracting total RNA from fish adipose tissues.
Background
Ribonucleic acids are genetic information carriers in biological cells and in parts of viruses and viroids. RNA is a long chain molecule formed by the condensation of ribonucleotides via phosphodiester bonds. One ribonucleotide molecule consists of a phosphate, a ribose and a base. RNA is a single strand formed by transcription by taking a strand of DNA as a template and using a base complementary pairing principle, has the main function of realizing the expression of genetic information on protein, and is a bridge in the process of transforming the genetic information into phenotype. In this process, the transfer RNA carries amino acid residues corresponding to the triplet codon to bind to the mRNA being translated, and then ribosomal RNA links the amino acid residues into a peptide chain via peptide bonds to form a protein molecule.
In the past, when RNA is extracted from animal body tissues, histocyte treatment is not in place, so that the RNA in fat cells cannot be completely dissolved in a dissolving solution, and a traditional extraction method usually only has one centrifugation treatment, so that the effective quantity of RNA extraction is greatly reduced.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for extracting total RNA from fish adipose tissues, which solves the problems that in the past, when RNA is extracted from animal body tissues, histocyte treatment is not in place, so that the RNA in adipose cells cannot be completely dissolved in a dissolving solution, and the traditional extraction method only has one centrifugation treatment, so that the effective quantity of RNA extraction is reduced.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for extracting total RNA from fish adipose tissues comprises the following steps:
step 1: preparation of clean working tool: thoroughly cleaning experimental tools such as a measuring cup, a measuring cylinder and the like to be used with distilled water, disinfecting the experimental tools, placing the experimental tools at a shady and ventilated position, and airing the experimental tools for later use;
step 2: basic treatment of fat cells: crushing and separating fresh adipose tissues, transferring the separated adipose tissues into a 1.5ml centrifuge tube by using a dropper, adding 1ml of Trizol, uniformly mixing, and standing for 5-15 minutes at room temperature;
and step 3: standard treatment before centrifugation: adding 0.2-0.5ml of chloroform into the standing centrifuge tube, oscillating for 15 seconds by using an oscillator, and standing for 2 minutes;
and 4, step 4: centrifugation of adipose tissue: placing the centrifugal tube in a refrigerator, when the temperature is reduced to 4 ℃, carrying out centrifugal operation on the centrifugal tube for 15 minutes, and then obtaining supernatant of the centrifuged layered liquid;
and 5: standard treatment before secondary centrifugation: adding 0.5ml-1.5 isopropanol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, and standing for 10 minutes at room temperature;
step 6: and (3) secondary centrifugal treatment: placing the liquid in a refrigerator, centrifuging the liquid for 10 minutes when the temperature is reduced to 4 ℃, and then pouring the supernatant of the layering solution;
and 7: and (3) carrying out three times of centrifugal treatment: adding 1ml of ethanol into the poured solution, slightly washing the precipitate, refrigerating the precipitate to 4 ℃, centrifuging the precipitate for 5 minutes, and pouring the supernatant of the layered solution;
and 8: air-drying to obtain RNA: and (3) drying the obtained product, and then adding an appropriate amount of DEPC water, heating to 65 ℃, and keeping for 10 minutes for dissolution.
Preferably, in step 2, the operator must wear gloves to perform the operation.
Preferably, in the step 7, the ethanol solution is a 75% solution.
Preferably, in the step 4, the liquid taking device and the related instruments are made of sterile disposable plastic products.
Preferably, in step 8, the DEPC must be operated in a fume hood.
Preferably, in the step 8, after the aqueous DEPC solution is used, the beaker containing the aqueous DEPC solution is sealed with aluminum foil and is steam-sterilized at high temperature and high pressure for 30 minutes.
Preferably, in the step 1, the glass product needs to be baked for 3 hours at 250 ℃.
(III) advantageous effects
The invention provides a method for extracting total RNA from fish adipose tissues. Compared with the prior art, the method has the following beneficial effects:
(1) the method for extracting total RNA from fish adipose tissues comprises the following steps of 2: basic treatment of fat cells: after fresh adipose tissues are crushed and separated, the separated adipose tissues are moved into a 1.5ml centrifuge tube by using a dropper, 1ml of Trizol is added, the mixture is uniformly mixed and kept stand for 5-15 minutes at room temperature, and by repeatedly treating the fish fat in the step 2, after the adipose tissues are physically crushed, the dropping tube is mixed with a reaction solvent, so that the decomposition of the adipose cells is effectively accelerated, and the problem that RNA in the adipose cells cannot be completely dissolved in a dissolving solution because the tissue cells are not completely treated when RNA is extracted from animal body tissues in the past is solved.
(2) The method for extracting total RNA from fish adipose tissues comprises the following steps of (4): centrifugation of adipose tissue: placing the centrifugal tube in a refrigerator, when the temperature is reduced to 4 ℃, carrying out centrifugal operation on the centrifugal tube for 15 minutes, and then obtaining the supernatant of the centrifugal post-lamination liquid, and the step 5: standard treatment before secondary centrifugation: adding 0.5ml-1.5 isopropanol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, standing for 10 minutes at room temperature, and carrying out step 7: and (3) carrying out three times of centrifugal treatment: adding 1ml of ethanol into the poured solution, slightly washing the precipitate, refrigerating the precipitate to 4 ℃, centrifuging for 5 minutes, pouring the supernatant of the layered solution, and performing combined treatment in the steps 4, 6 and 7 to completely crack and release RNA in the cells, thereby greatly increasing the RNA naturally released in the cells, and the device has a simple structure and is easy to maintain.
(3) The method for extracting total RNA from fish adipose tissues comprises the following steps of 3: standard treatment before centrifugation: adding 0.2-0.5ml of chloroform into the centrifuge tube after standing, shaking for 15 seconds by using a shaker, standing for 2 minutes, and performing step 5: standard treatment before secondary centrifugation: adding 0.5ml-1.5 isopropyl alcohol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, standing for 10 minutes at room temperature, and enabling the protective force generated before complete completion to be effectively kept in a single gear through the combined treatment setting in the step 3 and the step 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the attached tables in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to table 1, the embodiment of the present invention provides three technical solutions: a method for extracting total RNA from fish adipose tissues specifically comprises the following embodiments:
example 1
Step 1: preparation of clean working tool: thoroughly cleaning experimental tools such as a measuring cup, a measuring cylinder and the like to be used with distilled water, disinfecting the experimental tools, placing the experimental tools at a shady and ventilated position, and airing the experimental tools for later use;
step 2: basic treatment of fat cells: crushing and separating fresh adipose tissues, transferring the separated adipose tissues into a 1.5ml centrifuge tube by using a dropper, adding 1ml of Trizol, uniformly mixing, and standing for 5 minutes at room temperature;
and step 3: standard treatment before centrifugation: adding 0.2 chloroform into the standing centrifuge tube, oscillating for 15 seconds by using an oscillator, and standing for 2 minutes;
and 4, step 4: centrifugation of adipose tissue: placing the centrifugal tube in a refrigerator, when the temperature is reduced to 4 ℃, carrying out centrifugal operation on the centrifugal tube for 15 minutes, and then obtaining supernatant of the centrifuged layered liquid;
and 5: standard treatment before secondary centrifugation: adding 0.5ml of isopropanol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, and standing for 10 minutes at room temperature;
step 6: and (3) secondary centrifugal treatment: placing the liquid in a refrigerator, centrifuging the liquid for 10 minutes when the temperature is reduced to 4 ℃, and then pouring the supernatant of the layering solution;
and 7: and (3) carrying out three times of centrifugal treatment: adding 1ml of ethanol into the poured solution, slightly washing the precipitate, refrigerating the precipitate to 4 ℃, centrifuging the precipitate for 5 minutes, and pouring the supernatant of the layered solution;
and 8: air-drying to obtain RNA: and (3) drying the obtained product, and then adding an appropriate amount of DEPC water, heating to 65 ℃, and keeping for 10 minutes for dissolution.
Example 2
Step 1: preparation of clean working tool: thoroughly cleaning experimental tools such as a measuring cup, a measuring cylinder and the like to be used with distilled water, disinfecting the experimental tools, placing the experimental tools at a shady and ventilated position, and airing the experimental tools for later use;
step 2: basic treatment of fat cells: crushing and separating fresh adipose tissues, transferring the separated adipose tissues into a 1.5ml centrifuge tube by using a dropper, adding 1ml of Trizol, uniformly mixing, and standing for 10 minutes at room temperature;
and step 3: standard treatment before centrifugation: adding 0.3ml of chloroform into the standing centrifuge tube, oscillating for 15 seconds by using an oscillator, and standing for 2 minutes;
and 4, step 4: centrifugation of adipose tissue: placing the centrifugal tube in a refrigerator, when the temperature is reduced to 4 ℃, carrying out centrifugal operation on the centrifugal tube for 15 minutes, and then obtaining supernatant of the centrifuged layered liquid;
and 5: standard treatment before secondary centrifugation: adding 1.0 isopropanol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, and standing for 10 minutes at room temperature;
step 6: and (3) secondary centrifugal treatment: placing the liquid in a refrigerator, centrifuging the liquid for 10 minutes when the temperature is reduced to 4 ℃, and then pouring the supernatant of the layering solution;
and 7: and (3) carrying out three times of centrifugal treatment: adding 1ml of ethanol into the poured solution, slightly washing the precipitate, refrigerating the precipitate to 4 ℃, centrifuging the precipitate for 5 minutes, and pouring the supernatant of the layered solution;
and 8: air-drying to obtain RNA: and (3) drying the obtained product, and then adding an appropriate amount of DEPC water, heating to 65 ℃, and keeping for 10 minutes for dissolution.
Example 3
Step 1: preparation of clean working tool: thoroughly cleaning experimental tools such as a measuring cup, a measuring cylinder and the like to be used with distilled water, disinfecting the experimental tools, placing the experimental tools at a shady and ventilated position, and airing the experimental tools for later use;
step 2: basic treatment of fat cells: crushing and separating fresh adipose tissues, transferring the separated adipose tissues into a 1.5ml centrifuge tube by using a dropper, adding 1ml of Trizol, uniformly mixing, and standing for 15 minutes at room temperature;
and step 3: standard treatment before centrifugation: adding 0.5ml of chloroform into the standing centrifuge tube, oscillating for 15 seconds by using an oscillator, and standing for 2 minutes;
and 4, step 4: centrifugation of adipose tissue: placing the centrifugal tube in a refrigerator, when the temperature is reduced to 4 ℃, carrying out centrifugal operation on the centrifugal tube for 15 minutes, and then obtaining supernatant of the centrifuged layered liquid;
and 5: standard treatment before secondary centrifugation: adding 1.5 isopropanol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, and standing for 10 minutes at room temperature;
step 6: and (3) secondary centrifugal treatment: placing the liquid in a refrigerator, centrifuging the liquid for 10 minutes when the temperature is reduced to 4 ℃, and then pouring the supernatant of the layering solution;
and 7: and (3) carrying out three times of centrifugal treatment: adding 1ml of ethanol into the poured solution, slightly washing the precipitate, refrigerating the precipitate to 4 ℃, centrifuging the precipitate for 5 minutes, and pouring the supernatant of the layered solution;
and 8: air-drying to obtain RNA: and (3) drying the obtained product, and then adding an appropriate amount of DEPC water, heating to 65 ℃, and keeping for 10 minutes for dissolution.
Comparative experiment
According to claim 1, the existing manufacturer can operate the method for extracting total RNA from three fish adipose tissues, and compare the time and extraction amount of the total RNA extracted from the fish adipose tissues with the extraction amount and extraction time of the total RNA extracted from the fish adipose tissues, and it is shown that the extraction amount in the three examples is 44 μ l at the lowest, 9 μ l higher than the comparative example, 9.8 hours at the longest, and 6 hours shorter than the comparative example by laboratory tests.
Table 1: comparative example comparison table of extraction time and extraction amount
In step 2, an operator needs to wear gloves to operate, RNase enzyme is very stable, the RNase enzyme cannot be completely inactivated by a high-temperature high-pressure steam sterilization method and widely exists on the surface of human skin, in step 7, an ethanol solution is a 75% solution, the sterilization effect is the best, in step 4, a liquid taking device and related instruments are all sterile disposable plastic products, aseptic and pollution-free treatment is guaranteed, in step 8, DEPC is operated in a ventilation cabinet, DEPC is a virulent substance and is very strong in activity, in step 8, after the use of a DEPC aqueous solution is finished, a beaker containing the DEPC aqueous solution is sealed by an aluminum foil, high-temperature high-pressure steam sterilization is carried out for 30 minutes, the sterilization effect is guaranteed, and in step 1, the glass products need to be baked at 250 ℃ for 3 hours, and the sterilization effect is guaranteed.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for extracting total RNA from fish adipose tissues is characterized in that: the method comprises the following steps:
step 1: preparation of clean working tool: thoroughly cleaning experimental tools such as a measuring cup, a measuring cylinder and the like to be used with distilled water, disinfecting the experimental tools, placing the experimental tools at a shady and ventilated position, and airing the experimental tools for later use;
step 2: basic treatment of fat cells: crushing and separating fresh adipose tissues, transferring the separated adipose tissues into a 1.5ml centrifuge tube by using a dropper, adding 1ml of Trizol, uniformly mixing, and standing for 5-15 minutes at room temperature;
and step 3: standard treatment before centrifugation: adding 0.2-0.5ml of chloroform into the standing centrifuge tube, oscillating for 15 seconds by using an oscillator, and standing for 2 minutes;
and 4, step 4: centrifugation of adipose tissue: placing the centrifugal tube in a refrigerator, when the temperature is reduced to 4 ℃, carrying out centrifugal operation on the centrifugal tube for 15 minutes, and then obtaining supernatant of the centrifuged layered liquid;
and 5: standard treatment before secondary centrifugation: adding 0.5ml-1.5 isopropanol into the obtained supernatant, then shaking the mixed liquid gently and uniformly, and standing for 10 minutes at room temperature;
step 6: and (3) secondary centrifugal treatment: placing the liquid in a refrigerator, centrifuging the liquid for 10 minutes when the temperature is reduced to 4 ℃, and then pouring the supernatant of the layering solution;
and 7: and (3) carrying out three times of centrifugal treatment: adding 1ml of ethanol into the poured solution, slightly washing the precipitate, refrigerating the precipitate to 4 ℃, centrifuging the precipitate for 5 minutes, and pouring the supernatant of the layered solution;
and 8: air-drying to obtain RNA: and (3) drying the obtained product, and then adding an appropriate amount of DEPC water, heating to 65 ℃, and keeping for 10 minutes for dissolution.
2. The method of claim 1, wherein the total RNA is extracted from fish adipose tissue by the following steps: in step 2, the operator must wear gloves to perform the operation.
3. The method of claim 1, wherein the total RNA is extracted from fish adipose tissue by the following steps: in the step 7, the ethanol solution is 75% solution.
4. The method of claim 1, wherein the total RNA is extracted from fish adipose tissue by the following steps: in the step 4, the liquid taking device and related instruments are all made of sterile disposable plastic products.
5. The method of claim 1, wherein the total RNA is extracted from fish adipose tissue by the following steps: in said step 8, the DEPC has to be operated in a fume hood.
6. The method of claim 1, wherein the total RNA is extracted from fish adipose tissue by the following steps: in the step 8, after the DEPC aqueous solution is used, the beaker containing the DEPC aqueous solution is sealed by an aluminum foil and is sterilized by high-temperature high-pressure steam for 30 minutes.
7. The method of claim 1, wherein the total RNA is extracted from fish adipose tissue by the following steps: in the step 1, the glass product needs to be baked for 3 hours at 250 ℃.
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| CN202010146687.5A CN111172155A (en) | 2020-03-04 | 2020-03-04 | Method for extracting total RNA from fish adipose tissue |
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Citations (3)
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| CN107029237A (en) * | 2016-02-04 | 2017-08-11 | 康建胜 | Heat production enhancing compound enhancing norepinephrine class compound induces the application of brown fat cell heat production |
| CN107586775A (en) * | 2017-11-10 | 2018-01-16 | 信阳师范学院 | A kind of method that total serum IgE is extracted in the tissue from beef fat |
| CN108853503A (en) * | 2017-05-11 | 2018-11-23 | 中国科学院水生生物研究所 | Vitamin D receptor agonist is used to preventing and/or treating fat purposes |
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2020
- 2020-03-04 CN CN202010146687.5A patent/CN111172155A/en active Pending
Patent Citations (3)
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| CN107029237A (en) * | 2016-02-04 | 2017-08-11 | 康建胜 | Heat production enhancing compound enhancing norepinephrine class compound induces the application of brown fat cell heat production |
| CN108853503A (en) * | 2017-05-11 | 2018-11-23 | 中国科学院水生生物研究所 | Vitamin D receptor agonist is used to preventing and/or treating fat purposes |
| CN107586775A (en) * | 2017-11-10 | 2018-01-16 | 信阳师范学院 | A kind of method that total serum IgE is extracted in the tissue from beef fat |
Non-Patent Citations (5)
| Title |
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| TODORCEVIC, M等: "Gene expression profiles in Atlantic salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes", 《BMC GENOMICS》 * |
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Application publication date: 20200519 |