CN118325893A - A method for simultaneously extracting total metabolites, total RNA, DNA and proteins from biological tissues - Google Patents

Abstract

The invention discloses a method for simultaneously extracting total metabolites, total RNA, DNA and proteins in biological tissues, which comprises the steps of firstly, using a methanol/chloroform mixture to perform initial metabolite extraction, and simultaneously keeping the RNA, the DNA and the proteins in a precipitate; then by adding TRIzol reagent and chloroform, the effective separation of RNA, DNA and protein is realized by utilizing the distribution characteristics of different biomolecules in different phases. The method optimizes the traditional extraction process, and by accurately controlling the addition amount of reagent and centrifugation conditions, the sample loss is reduced to the maximum extent and the quality of the extract is ensured. The method is simple, convenient and effective, solves the problems that each biomolecule is extracted independently to cause pollution, the extraction result is inaccurate, the extraction process is low in efficiency and the like, can obtain high-quality products, and meets the requirements of subsequent experiments.

Description

A method for simultaneously extracting total metabolites, total RNA, DNA and proteins from biological tissues

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a method for simultaneously extracting total metabolites, total RNA, DNA and proteins in biological tissues.

Background technique

At present, researchers have gained a deep understanding of biological characteristics such as gene function, expression regulation, protein structure and function by isolating and studying total metabolites, DNA, RNA, proteins and other biomolecules in tissues, thereby revealing the mechanisms of various biological processes in organisms. Secondly, the extraction and separation of biomolecules in biological samples also provides a basis for disease research and diagnosis. By analyzing specific biomolecules in samples such as blood and tissues, researchers can accurately diagnose diseases, monitor disease progression, and evaluate treatment plans, providing important support for progress in the medical field. In addition, the extraction and separation of biomolecules in biological samples is also crucial for drug development and evaluation, bioengineering and biomanufacturing, and ecological research and environmental monitoring. In the process of drug development, understanding the metabolic process, targets and side effects of drugs in organisms requires the separation and study of biomolecules; in the field of ecology and environmental science, analyzing molecules such as DNA and RNA in biological samples can assess the health of ecosystems, biodiversity, and the distribution and impact of environmental pollutants, providing data support for environmental protection.

Traditionally, biological research often requires the use of multiple different experimental methods to separately extract and separate various single biomolecules in biological samples, such as metabolites, RNA, DNA, and proteins. These traditional methods usually need to be performed separately, and may cause sample loss or contamination during processing, which is not only time-consuming but also may lead to reduced comparability between samples, thereby affecting the accuracy and reproducibility of the results. In addition, various "omics" technologies are currently used to fully understand the complex interactions of biomolecules within biological systems. The technological development of genomics, transcriptomics, proteomics, and metabolomics enables the monitoring and quantification of biomolecules in a high-throughput manner. The integration of omics technologies is difficult when the same samples are not used for comprehensive molecular analysis.

Summary of the invention

The object of the present invention is to provide a method for simultaneously extracting total metabolites, total RNA, DNA and protein in biological tissues, which method can improve the extraction efficiency and the accuracy of the extraction results.

In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

A method for simultaneously extracting total metabolites, total RNA, DNA and proteins from biological tissues, comprising the following steps:

A. Take fresh tissue and place it in a centrifuge tube, and grind it into fine powder in a liquid nitrogen environment;

B. Adding a mixture of methanol and chloroform to the centrifuge tube in step A, rotating and incubating to precipitate metabolites, centrifuging to obtain a supernatant and a precipitate, wherein the metabolites are precipitated and retained in the supernatant, and the supernatant is transferred to a new centrifuge tube and stored at low temperature; RNA, DNA and protein are retained in the precipitate, and RNA, DNA and protein are continuously separated from the precipitate in sequence;

C. Add TRIzol reagent to the precipitate obtained in step B to obtain a cell lysate, incubate the cell lysate and centrifuge to obtain a supernatant and a precipitate, and discard the precipitate;

D. Transfer the supernatant obtained in step C to a new centrifuge tube, add chloroform, shake vigorously to mix and incubate, and centrifuge to obtain an aqueous phase, an intermediate phase, and a phenol-chloroform phase;

E. The RNA is retained in the aqueous phase in step D. The aqueous phase is transferred to a new centrifuge tube, isopropanol is added, the mixture is mixed by inversion, and then placed in a refrigerator for standing. The supernatant and precipitate are obtained by centrifugation. The supernatant is discarded, and the precipitate is washed multiple times. The washed precipitate is centrifuged again and the residual liquid is removed by aspiration. The precipitate is dried and dissolved in water for storage at low temperature.

F. DNA is retained in the intermediate phase in step D, and protein is retained in the phenol-chloroform phase in step D. Ethanol is added to the mixture of the phenol-chloroform phase and the intermediate phase, and the mixture is inverted and allowed to stand, and centrifuged to obtain a precipitate and a supernatant;

G. The DNA is retained in the precipitate obtained in step F, and the precipitate is transferred to a new centrifuge tube, and the precipitate is washed multiple times, ethanol is added to the precipitate, and the precipitate is allowed to stand, centrifuged and the supernatant is discarded, and the precipitate is dried and dissolved in water and stored at low temperature;

H. The protein is retained in the supernatant obtained in step F. The supernatant is transferred to a test tube, isopropanol is added, the mixture is allowed to stand, and a precipitate and a supernatant are obtained after centrifugation. The supernatant is discarded, and the protein is retained in the precipitate. The precipitate is washed multiple times, dried after centrifugation, and sodium dodecyl sulfate is added to a water bath to completely dissolve the precipitate, and then stored at low temperature.

Furthermore, the specific steps of step A are: taking the target animal under cryo-anesthesia under low temperature conditions, then taking the tissue and placing it in a centrifuge tube, and immediately freezing it in liquid nitrogen; adding liquid nitrogen to the centrifuge tube containing the tissue to embrittle the tissue, and grinding the tissue into powder with a homogenizer until no obvious particles are present.

Preferably, in step B, the volume ratio between methanol and chloroform is 9:1;

The rotation temperature is 4°C, the rotation time is 10 min, and the rotation speed is 50 rpm; the centrifugal temperature is 4°C, the centrifugal time is 10 min, and the centrifugal speed is 500g; and the low temperature is -80°C.

Preferably, the volume ratio of the TRIzol reagent in step C to the chloroform in step D is 1:0.2; in step C, the incubation temperature is room temperature, and the incubation time is 15 min; the centrifugation temperature is 4°C, the centrifugation time is 10 min, and the centrifugation speed is 13000 g.

Preferably, in step D, the incubation temperature is room temperature, and the incubation time is 3 min; the centrifugation temperature is 4°C, the centrifugation time is 15 min, and the centrifugation speed is 13000 g.

Preferably, in step E, the volume ratio between the isopropanol and the aqueous phase is 1:1, the standing temperature is -80°C, and the standing time is 1h; the centrifugation temperature is 4°C, the centrifugation time is 10min, and the centrifugation speed is 13000g; the re-centrifugation temperature is 4°C, the centrifugation time is 5min, and the centrifugation speed is 13000g;

The washing method is specifically as follows: adding a washing solution DEPC-75% ethanol to the precipitate, wherein the DEPC-75% ethanol is an ethanol solution with a volume fraction of 75% prepared by using DEPC water, and suspending the RNA precipitate by turning it upside down for washing, centrifuging at a centrifugal temperature of 4° C., a centrifugal time of 5 min, and a centrifugal speed of 7500 g, discarding the supernatant, and repeating the washing process once; the volume ratio between the washing solution DEPC-75% ethanol and the TRIzol reagent added each time is 1:1;

The precipitation is dried by opening the lid and drying at room temperature for 5 to 10 minutes; the water dissolved in the water after the precipitation is dried is RNase-free double distilled water; and the low temperature is -80°C.

Preferably, in step F, the volume ratio of the mixture of the phenol-chloroform phase and the intermediate phase, the TRIzol reagent and ethanol is 1:1:0.3; the volume fraction of ethanol is 100%; the standing temperature is room temperature, and the standing time is 3 minutes; the centrifugation temperature is 4°C, the centrifugation time is 5 minutes, and the centrifugation speed is 7500g.

Preferably, in step G, the washing method is specifically as follows: adding 10% ethanol as a washing liquid to the precipitate, standing at room temperature for 10 to 30 minutes, centrifuging at a centrifugal temperature of 4°C, a centrifugal speed of 7500g, and a centrifugal time of 5 minutes, discarding the supernatant, and repeating the washing process once;

After the precipitate is washed, 75% ethanol by volume is added again; the standing temperature is room temperature, and the standing time is 10 to 30 minutes; the centrifugation temperature is 4°C, the centrifugation time is 5 minutes, and the centrifugation speed is 4000g; the precipitate is dried at room temperature with the lid opened for 5 to 10 minutes; the water dissolved in the water after the precipitate is dried is RNase-free double distilled water; the volume ratio of TRIzol reagent, 10% ethanol by volume and 75% ethanol by volume is 1:1:1.

Preferably, in step H, the standing temperature is room temperature, the standing time is 10 min; the centrifugation temperature is 4° C., the centrifugation time is 10 min, the centrifugation speed is 12000 g; the volume ratio of TRIzol reagent to isopropanol is 1:1.5;

The washing method is specifically as follows: adding a washing solution 0.3M guanidine hydrochloride-95% ethanol solution to the precipitate, wherein the 0.3M guanidine hydrochloride-95% ethanol solution is an ethanol solution with a volume fraction of 95% prepared by using 0.3M guanidine hydrochloride, standing at room temperature for 10 minutes, centrifuging at a centrifugal temperature of 4°C, a centrifugal speed of 7500g, and a centrifugal time of 5 minutes, and discarding the supernatant; adding anhydrous ethanol to the precipitate, standing at room temperature for 10 minutes, centrifuging at a centrifugal temperature of 4°C, a centrifugal speed of 7500g, and a centrifugal time of 5 minutes, and discarding the supernatant; the volume ratio of TRIzol reagent, washing solution 0.3M guanidine hydrochloride-95% ethanol and anhydrous ethanol is 1:1:1;

The drying is performed by opening the cover and drying at room temperature for 20 to 30 minutes; the mass fraction of the sodium dodecyl sulfate is 1%, and the water bath temperature is 50 to 60°C.

Preferably, the centrifuge tubes are all RNase-free centrifuge tubes.

The present invention uses a series of operations to ensure that these biomacromolecules are extracted simultaneously from a single tissue sample effectively and with high purity. The key steps include the use of a methanol/chloroform mixture for initial metabolite extraction, which simultaneously leaves RNA, DNA, and protein in the precipitate; then, by adding TRIzol reagent and chloroform, the distribution characteristics of different biomolecules in different phases are utilized to achieve effective separation of RNA, DNA, and protein. This method optimizes the traditional extraction process, and by precisely controlling the amount of reagent added and the centrifugation conditions, it minimizes sample loss while ensuring the quality of the extract. In addition, the method also places special emphasis on the details of the sample preparation process, such as tissue freezing and grinding under low temperature conditions, to ensure the integrity and stability of the biomolecules. This series of operations is designed to meet the strict requirements for high efficiency and high purity of sample processing in multi-omics research, providing reliable sample preparation for subsequent analysis.

The beneficial effects of the present invention are that it is simple, convenient and effective, and solves the problems that the individual extraction of each biological molecule may cause contamination, inaccurate extraction results and low efficiency of the extraction process, and can obtain high-quality products to meet the requirements of subsequent experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a flow chart of the present invention;

Figure 2 is a chromatogram of tissue metabolites; (a) is a chromatogram of metabolites in the brain tissue of female rats, (b) is a chromatogram of metabolites in the brain tissue of male rats; (c) is a chromatogram of metabolites in the muscle tissue of female crickets; (d) is a chromatogram of metabolites in the muscle tissue of male crickets;

FIG3 is a graph showing the results of 1% agarose gel electrophoresis of the integrity of total RNA extracted from mouse brain tissue and cricket muscle tissue in Example 1 and Example 2, respectively, wherein: F-MB is total RNA from female mouse brain tissue, M-MB is total RNA from male mouse brain tissue, F-GL is total RNA from female cricket muscle tissue, and M-GL is total RNA from male cricket muscle tissue;

FIG4 is an electrophoresis diagram of the detection of the PCR amplification results of the COI gene by the mouse brain tissue and cricket muscle tissue DNA extracted in Example 1 and Example 2, respectively, in which: F-MB is the PCR amplification result of the COI gene in the female mouse brain tissue, F-GL is the PCR amplification result of the COI gene in the female cricket muscle tissue, M-MB is the PCR amplification result of the COI gene in the male mouse brain tissue, and M-GL is the PCR amplification result of the COI gene in the male cricket muscle tissue; Marker is the DNA standard molecular scalar D1000 DNA Ladder strip;

Figure 5 is a Western Blot result diagram of the total protein extracted from mouse brain tissue and cricket muscle tissue in Example 1 and Example 2, respectively; in the figure: F-MB is the total protein of female mouse brain tissue, F-GL is the total protein of female cricket muscle tissue, M-MB is the total protein of male mouse brain tissue, and M-GL is the total protein of male cricket muscle tissue.

Detailed ways

The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

In the following examples, the reagents required for the examples are as follows:

TRIzol ^TM Reagent (Thermo Fisher Scientific, USA),

Chloroform (Sinopharm Chemical Reagent Co., Ltd.),

Isopropyl alcohol (Sinopharm Chemical Reagent Co., Ltd.),

75% anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.),

Agarose (BBI Life Sciences Corporation),

Gel Red Nucleic Acid Dye (BBI Life Sciences Corporation),

DNALadder (Tiangen Biochemical Technology Co., Ltd.),

RNase-free double distilled water (Shanghai Sangon Biotechnology Co., Ltd.),

10×MOPS buffer (Beijing Solebow Technology Co., Ltd.),

Methanol (Sinopharm Chemical Reagent Co., Ltd.),

Guanidine hydrochloride (Shanghai Sangon Biotechnology Co., Ltd.),

Sodium dodecyl sulfate (Shanghai Sangon Biotechnology Co., Ltd.).

In the following examples, the instruments required for the examples are as follows:

5424R refrigerated centrifuge (Eppendorf, Germany),

JA2003 balance (Tianjin Tianma Hengji Instrument Co., Ltd.),

Chemidoc XRS+ gel imaging system (BIO-RAD, USA),

ND2000 NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

High-speed electric tissue grinder (OSE-Y30) (Tiangen Biochemical Technology (Beijing) Co., Ltd.),

Votex oscillator (GENIE, Germany),

Electrophoresis instrument (BIO-RAD, USA),

Pipette (Eppendorf, Germany),

Ophthalmic forceps (Shanghai Sangon Biotechnology Co., Ltd.),

Ophthalmic scissors (Shanghai Sangon Biotechnology Co., Ltd.).

In the following examples, the hippocampus tissue of mouse brain and the tissue of male and female adult two-spotted crickets were used as materials for the following description; mice were purchased from Nanjing Jicui Yaokang Biological Co., Ltd.

Example 1

As shown in FIG1 , a method for simultaneously extracting total metabolites, total RNA, DNA and protein from biological tissues comprises the following steps:

A. Take about 30 mg of hippocampal tissue from C57 mice and place it in a 1.5 mL RNase-free centrifuge tube, and immediately freeze it in liquid nitrogen; add 1 mL of liquid nitrogen to the centrifuge tube containing the tissue to embrittle the tissue, and grind the tissue into powder using a TGrinder third-generation high-speed tissue grinder until no obvious particles are present;

B. Add a mixture of methanol and chloroform in a volume ratio of 9:1 to the centrifuge tube in step A, rotate and incubate to precipitate metabolites, the rotation temperature is 4°C, the rotation time is 10 min, the rotation speed is 50 rpm, and centrifuge to obtain a supernatant and a precipitate, the centrifugation temperature is 4°C, the centrifugation time is 10 min, and the centrifugation speed is 500g. The metabolites are precipitated and retained in the supernatant, and the supernatant is transferred to a new 1.5 mL RNase-free centrifuge tube and stored at -80°C or the extracted metabolites are analyzed by mass spectrometry to obtain a series of separated peaks, reflecting the abundance and characteristics of different metabolites, as shown in Figure 2; RNA, DNA and protein are retained in the precipitate, and RNA, DNA and protein are continuously separated from the precipitate in sequence;

C. Add 1 mL of TRIzol reagent to the precipitate obtained in step B to obtain a cell lysate, incubate the cell lysate at room temperature for 15 min, and then centrifuge at a temperature of 4° C., a time of 10 min, and a speed of 13000 g to obtain a supernatant and a precipitate, and discard the precipitate;

D. Transfer the supernatant obtained in step C to a new 1.5 mL RNase-free centrifuge tube, add 200 uL chloroform, shake vigorously and mix, incubate at room temperature for 3 min, and centrifuge to obtain an aqueous phase, an intermediate phase, and a phenol-chloroform phase. The centrifugation temperature is 4 ° C, the centrifugation time is 15 min, and the centrifugation speed is 13000 g;

E. The RNA is retained in the aqueous phase in step D. The aqueous phase is transferred to a new 1.5 mL RNase-free centrifuge tube, and an equal volume of isopropanol is added to the aqueous phase. The tube is mixed by inversion and then placed in a refrigerator for standing at -80°C for 1 h. The supernatant and precipitate are obtained by centrifugation at 4°C for 10 min and at a speed of 13000 g. The supernatant is discarded, and the precipitate is washed multiple times. The washing method is as follows: 1 mL of washing solution DEPC-75% ethanol is added to the precipitate. DEPC-75% ethanol is an ethanol solution with a volume fraction of 75% prepared with DEPC water. The RNA precipitate is suspended and washed by inversion. The centrifugation temperature is 4°C and the centrifugation time is 10 min. The supernatant was discarded after centrifugation under the conditions of 5 minutes and a centrifugal speed of 7500g, and the washing process was repeated once; the washed precipitate was centrifuged again and the residual liquid was sucked off, and the re-centrifugation temperature was 4°C, the centrifugal time was 5 minutes, and the centrifugal speed was 13000g; the precipitate was opened and dried at room temperature for 5-10 minutes, and then dissolved in 30-50uL of RNase-free double distilled water and stored at -80°C. The subsequent RNA electrophoresis analysis showed that the RNA electrophoresis bands extracted from the hippocampus tissue of the male and female mice were clear and complete, without mixed bands and DNA contamination, indicating that the RNA integrity was good (Figure 3);

F. DNA is retained in the intermediate phase in step D, and protein is retained in the phenol-chloroform phase in step D. 300uL of anhydrous ethanol is added to the mixture of the phenol-chloroform phase and the intermediate phase, and the mixture is inverted and allowed to stand at room temperature for 3 minutes. After centrifugation, a precipitate and a supernatant are obtained. The centrifugation temperature is 4°C, the centrifugation time is 5 minutes, and the centrifugation speed is 7500g.

G. DNA is retained in the precipitate obtained in step F, and the precipitate is transferred to a new centrifuge tube, and the precipitate is washed several times, 1 mL of washing liquid with a volume fraction of 10% ethanol is added to the precipitate, and the precipitate is allowed to stand at room temperature for 10 to 30 minutes, and the supernatant is discarded under the conditions of a centrifugal temperature of 4°C, a centrifugal speed of 7500g, and a centrifugal time of 5 minutes, and the washing process is repeated once; 1 mL of 75% ethanol by volume is added to the precipitate, and the precipitate is allowed to stand at room temperature for 10 to 30 minutes, and the supernatant is discarded by centrifugation, wherein the centrifugal temperature is 4°C, the centrifugal time is 5 minutes, and the centrifugal speed is 4000g; the precipitate is opened and dried at room temperature for 5 to 10 minutes, and then dissolved in 30-50uL of RNase-free double distilled water and stored at -80°C; then the DNA extracted from the hippocampus tissue of the male and female mice is amplified with the universal primer COI, and the COI electrophoresis band is clear and complete, without any mixed bands, and without DNA contamination, indicating that the DNA meets the requirements of subsequent experiments (Figure 4);

H. The protein is retained in the supernatant obtained in step F. The supernatant is transferred to a 5 mL RNase-free test tube, 1.5 mL of isopropanol is added, and the mixture is allowed to stand at room temperature for 10 min. After centrifugation, a precipitate and a supernatant are obtained. The centrifugation temperature is 4° C., the centrifugation time is 10 min, and the centrifugation speed is 12000 g. The supernatant is discarded, and the protein is retained in the precipitate. The precipitate is washed multiple times. The washing method is specifically as follows: 1 mL of a washing solution 0.3 M guanidine hydrochloride-95% ethanol solution is added to the precipitate. The 0.3 M guanidine hydrochloride-95% ethanol solution is an ethanol solution with a volume fraction of 95% prepared using 0.3 M guanidine hydrochloride, and the mixture is allowed to stand at room temperature for 10 min. Under the conditions of centrifugal temperature of 4°C, centrifugal speed of 7500g and centrifugal time of 5min, the supernatant was discarded after centrifugation, 1mL of anhydrous ethanol was added to the precipitate, and it was allowed to stand at room temperature for 10min. After centrifugation at a centrifugal temperature of 4°C, a centrifugal speed of 7500g and centrifugal time of 5min, the supernatant was discarded after centrifugation, and the lid was opened and dried at room temperature for 20-30min. 40-60uL of 1% sodium dodecyl sulfate was added and the precipitate was completely dissolved in a water bath at a temperature of 50-60°C and then stored at low temperature. The total protein bands obtained after subsequent SDS-PAGE electrophoresis were clearly distinguishable with clear boundaries and no blurring or diffusion (Figure 5).

Example 2

The method steps in this example are consistent with those in Example 1, except that the C57 mice in step A are replaced with two-spotted cricket adults, and the two-spotted cricket nymphs are purchased from the Henan Cricket King Ecological Breeding Base and then raised in the laboratory. Two-spotted cricket adults (2-3 weeks) are taken and cryoanaesthetized for 5-7 minutes at -20°C low temperature, and then 40-50 mg of tissue is taken with small medical ophthalmic forceps and scissors, both of which are 12.5 cm in length. The other method steps are consistent with those in Example 1, and the corresponding results are shown in Figures 2-5.

As shown in Figure 3, the electrophoresis bands are clear and complete, without any mixed bands or DNA contamination, indicating that the RNA extracted from the cricket muscle tissue has good integrity.

As shown in Figure 4, the electrophoresis bands are clear and single and consistent with the molecular weight of the COI gene, indicating that the DNA extracted from the cricket muscle tissue has a good purity.

As shown in Figure 5, the SDS-PAGE protein band is single and dark in color, indicating that the concentration of the extracted protein is high.

Claims (10)

1. A method for simultaneously extracting total metabolites, total RNA, DNA and proteins in biological tissues, comprising the steps of:

A. placing fresh tissues into a centrifuge tube, and fully grinding the fresh tissues into fine powder in a liquid nitrogen environment;

B. Adding a mixed solution of methanol and chloroform into the centrifuge tube in the step A, rotationally incubating to separate out metabolites, centrifuging to obtain supernatant and precipitate, separating out the metabolites, reserving the supernatant, transferring the supernatant into a new centrifuge tube, and preserving at low temperature; RNA, DNA and protein remain in the precipitate, from which RNA, DNA and protein are successively separated in succession;

C. adding TRIzol reagent into the precipitate obtained in the step B to obtain a cell lysate, incubating the cell lysate, centrifuging to obtain a supernatant and a precipitate, and discarding the precipitate;

D. c, transferring the supernatant obtained in the step C into a new centrifuge tube, adding chloroform, shaking vigorously, mixing and incubating, and centrifuging to obtain a water phase, an intermediate phase and a phenol-chloroform phase;

E. The RNA is reserved in the water phase in the step D, the water phase is transferred to a new centrifuge tube, isopropanol is added, the mixture is reversed and mixed uniformly, the mixture is placed in a refrigerator for standing, the supernatant and the sediment are obtained through centrifugation, the supernatant is discarded, the sediment is washed for multiple times, the washed sediment is centrifuged again, residual liquid is sucked, and the sediment is dissolved in water for low-temperature storage after being dried;

F. DNA is reserved in the intermediate phase in the step D, protein is reserved in the phenol-chloroform phase in the step D, ethanol is added into the mixture of the phenol-chloroform phase and the intermediate phase, the mixture is reversed and kept stand, and precipitation and supernatant are obtained after centrifugation;

G. F, keeping DNA in the sediment obtained in the step F, transferring the sediment into a new centrifuge tube, washing the sediment for multiple times, adding ethanol into the sediment, standing, centrifuging, discarding supernatant, drying the sediment, and then dissolving in water for low-temperature storage;

H. And F, reserving protein in the supernatant obtained in the step F, transferring the supernatant into a test tube, adding isopropanol, standing, centrifuging to obtain precipitate and supernatant, discarding the supernatant, reserving the protein in the precipitate, washing the precipitate for multiple times, centrifuging, drying, adding sodium dodecyl sulfate into the solution to completely dissolve the precipitate, and preserving at a low temperature.

2. The method for simultaneously extracting total metabolites, total RNA, DNA and protein of biological tissue according to claim 1, wherein the specific steps of the step A are as follows: taking the raised target animal, carrying out freezing anesthesia under the low-temperature condition, then taking the tissue, placing the tissue into a centrifuge tube, and immediately placing the tissue into liquid nitrogen for freezing; the tissue was embrittled by adding liquid nitrogen to a centrifuge tube containing the tissue, and the tissue was ground to a powder with a homogenizer until no distinct particles were present.

3. A method for simultaneous extraction of total metabolites, total RNA, DNA and proteins in biological tissue according to claim 1 or 2, wherein in step B, the volume ratio between methanol and chloroform is 9:1;

The rotation temperature is 4 ℃, the rotation time is 10min, and the rotation speed is 50rpm; the centrifugal temperature is 4 ℃, the centrifugal time is 10min, and the centrifugal rotating speed is 500g; the low temperature is-80 ℃.

4. A method for simultaneous extraction of total metabolites, total RNA, DNA and proteins in biological tissue according to claim 1 or 2, wherein the volume ratio between TRIzol reagent in step C and chloroform in step D is 1:0.2; in the step C, the incubation temperature is room temperature, and the incubation time is 15min; the centrifugation temperature is 4 ℃, the centrifugation time is 10min, and the centrifugation rotating speed is 13000g.

5. A method for simultaneous extraction of total metabolites, total RNA, DNA and proteins in biological tissue according to claim 1 or 2, wherein in step D, the incubation temperature is room temperature and the incubation time is 3min; the centrifugation temperature is 4 ℃, the centrifugation time is 15min, and the centrifugation rotating speed is 13000g.

6. The method for simultaneous extraction of total metabolites, total RNA, DNA and protein of biological tissue according to claim 1 or 2, wherein in step E, the volume ratio between isopropyl alcohol and aqueous phase is 1:1, the resting temperature is-80 ℃, the resting time is 1h; the centrifugal temperature is 4 ℃, the centrifugal time is 10min, and the centrifugal rotating speed is 13000g; the re-centrifugation temperature is 4 ℃, the centrifugation time is 5min, and the centrifugation rotating speed is 13000g;

The washing method specifically comprises the following steps: adding washing liquid DEPC-75% ethanol into the precipitate, wherein the DEPC-75% ethanol is ethanol solution with the volume fraction of 75% prepared by adopting DEPC water, suspending the RNA precipitate upside down for washing, centrifuging at the centrifugation temperature of 4 ℃ for 5min and the centrifugation speed of 7500g, discarding the supernatant, and repeating the washing process for one time; the volume ratio of the washing liquid DEPC-75% ethanol to the TRIzol reagent added each time is 1:1;

the precipitation drying is that the cover is opened and the room temperature is dried for 5 to 10 minutes; the water dissolved in the water after the precipitate is dried is RNase-free double distilled water; the low temperature is-80 ℃.

7. The method for simultaneous extraction of total metabolites, total RNA, DNA and proteins in biological tissue according to claim 1 or 2, wherein in step F, the volume ratio between the mixture of phenol-chloroform phase and mesophase, TRIzol reagent and ethanol is 1:1:0.3; ethanol volume fraction 100%; the standing temperature is room temperature, and the standing time is 3min; the centrifugation temperature is 4 ℃, the centrifugation time is 5min, and the centrifugation rotating speed is 7500g.

8. A method for simultaneous extraction of total metabolites, total RNA, DNA and proteins in biological tissue according to claim 1 or 2, wherein in step G, the washing method is specifically: adding ethanol with the volume fraction of 10% into the precipitate, standing at room temperature for 10-30 min, centrifuging at 4 ℃ and at a centrifugal speed of 7500g for 5min, discarding the supernatant, and repeating the washing process once;

Adding ethanol with the volume fraction of 75% again after the precipitation washing is finished; the standing temperature is room temperature, and the standing time is 10-30 min; the centrifugation temperature is 4 ℃, the centrifugation time is 5min, and the centrifugation rotating speed is 4000g; the precipitation drying is that the cover is opened and the room temperature is dried for 5 to 10 minutes; the water dissolved in the water after the precipitation is dried is RNase-free double distilled water; the volume ratio of TRIzol reagent, 10% ethanol by volume, and 75% ethanol by volume was 1:1:1.

9. The method for simultaneous extraction of total metabolites, total RNA, DNA and protein of biological tissue according to claim 1 or 2, wherein in step H, said resting temperature is room temperature and the resting time is 10min; the centrifugal temperature is 4 ℃, the centrifugal time is 10min, and the centrifugal rotating speed is 12000g; the volume ratio of TRIzol reagent to isopropanol is 1:1.5;

The washing method specifically comprises the following steps: adding 0.3M guanidine hydrochloride-95% ethanol solution into the precipitate, wherein the 0.3M guanidine hydrochloride-95% ethanol solution is 95% ethanol solution prepared by 0.3M guanidine hydrochloride, standing at room temperature for 10min, centrifuging to remove supernatant under the conditions of a centrifuging temperature of 4 ℃ and a centrifuging speed of 7500g and a centrifuging time of 5min, adding absolute ethanol into the precipitate, standing at room temperature for 10min, centrifuging to remove supernatant under the conditions of a centrifuging temperature of 4 ℃ and a centrifuging speed of 7500g and a centrifuging time of 5 min; TRIzol reagent, washing solution 0.3M guanidine hydrochloride-95% ethanol and absolute ethanol in a volume ratio of 1:1:1;

The drying is that the cover is opened and the room temperature is dried for 20 to 30 minutes; the mass fraction of the sodium dodecyl sulfate is 1%, and the water bath temperature is 50-60 ℃.

10. The method for simultaneously extracting total metabolites, total RNA, DNA and protein of biological tissue according to claim 1 or 2, wherein said centrifuge tubes are RNase-free centrifuge tubes.