Method for identifying RNA (Ribonucleic Acid) binding protein in intramuscular fat cells or subcutaneous fat cells of pigs

Abstract

The invention belongs to the field of biology, and discloses a method for identifying RNA (Ribonucleic Acid) binding protein in porcine intramuscular fat cells or subcutaneous fat cells, which comprises the following steps: step 1, taking porcine muscle tissues or subcutaneous fat tissues to carry out meat mashing and digestion, and collecting mature intramuscular fat cells or subcutaneous fat cells; step 2, washing intramuscular fat cells or subcutaneous fat cells, and carrying out cross-linking treatment by adopting ultraviolet irradiation; step 3, collecting intramuscular fat cells or subcutaneous fat cells after cross-linking treatment, and performing lysis under the action of a cell lysis solution to obtain a lysis solution; step 4, adding magnetic beads into the lysate, and carrying out repeated resuspension and elution to obtain an eluent; and 5, separating the RNA binding protein from the magnetic beads, and incubating. According to the method, intramuscular fat cells or subcutaneous fat cells are treated by adopting a UV cross-linking solidification method, and then RNA binding protein is captured through magnetic beads, so that the RNA binding protein is successfully obtained.

Description

Technical Field

[0001] The present invention relates to the field of biology, and specifically to a method for identifying RNA-binding proteins in porcine intramuscular adipocytes or subcutaneous adipocytes. Background Art

[0002] Intramuscular fat content is a key factor determining pork quality, affecting meat flavor, juiciness, tenderness, color, etc. Therefore, in-depth research and revelation of the regulatory mechanism of porcine intramuscular fat deposition are of great significance for increasing intramuscular fat content and improving meat quality. It has been found that due to the particularity of the location of intramuscular adipocytes, their differentiation process is affected by various factors in the muscle microenvironment, and there are differences in the regulatory mechanisms of subcutaneous adipocyte differentiation. However, scientists at home and abroad have not been able to make major breakthroughs in the research on the key regulatory molecules of porcine intramuscular fat deposition and their differential regulatory mechanisms. One of the reasons is that the current research on the regulatory mechanism of intramuscular fat deposition mainly focuses on transcriptional regulation, signal transduction, and epigenetics, etc., but there is a lack of research on the role and mechanism of post-transcriptional regulation mediated by RNA-binding proteins in intramuscular fat deposition.

[0003] RNA-binding proteins are a class of key post-transcriptional regulatory factors, and their regulation of RNA fate is the main pathway for gene expression regulation during growth and development. Research has shown that RNA-binding proteins regulate RNA metabolic processes, including RNA stability, translation, alternative splicing, and localization, by binding to target RNAs in cells.

[0004] Regarding the separation research of RNA-binding proteins, the following techniques can be referred to:

[0005] Publication No. CN 116875668 A, with the theme of a method for capturing RNA-RNA interactions mediated by specific RNA-binding proteins, discloses that by UV cross-linking to fix the interaction between RNA-binding proteins and RNAs in cells, after lysing the cells, specific RNA-binding proteins and their bound RNAs are enriched on magnetic beads by magnetic beads conjugated with specific antibodies, the RNAs are cleaved into fragments, and then pCp-biotin is labeled at the 3' end of the RNAs and proximal ligation is performed on the magnetic beads.

[0006] Using UV cross-linking to fix RNA-binding proteins and the RNAs bound to these binding proteins is a relatively simple and feasible technique for separating RNA-binding proteins. In the above scheme, the cells used are HeLa cells.

[0007] The above scheme still has certain limitations in the extraction and separation of RNA-binding proteins in tissues, especially in the separation and extraction of RNA-binding proteins in intramuscular fat, where there are significant difficulties.

[0008] Recent studies have shown that RNA-binding proteins play an important regulatory role in fat deposition. However, no RNA-binding proteins that specifically regulate intramuscular fat deposition in pigs have been reported. Summary of the Invention

[0009] The object of the present invention is to provide a method for identifying RNA-binding proteins in pig intramuscular adipocytes or subcutaneous adipocytes. The method uses a UV cross-linking and curing method to treat intramuscular adipocytes or subcutaneous adipocytes, and then uses magnetic beads to capture RNA-binding proteins to successfully obtain RNA-binding proteins.

[0010] The present invention discloses a method for identifying RNA-binding proteins in pig intramuscular adipocytes or subcutaneous adipocytes, comprising the following steps:

[0011] Step 1: Muscle tissue or subcutaneous fat tissue is minced and digested to collect intramuscular fat cells or subcutaneous fat tissue;

[0012] Step 2: washing the intramuscular adipocytes or subcutaneous adipose tissue and then subjecting them to ultraviolet light cross-linking treatment;

[0013] Step 3: collecting the cross-linked intramuscular adipocytes or subcutaneous adipose tissue, and lysing them with a cell lysate to obtain a lysate;

[0014] Step 4: Add magnetic beads to the lysate, resuspend and elute multiple times to obtain an eluate;

[0015] Step 5: Separate the RNA-binding proteins from the magnetic beads and incubate.

[0016] In the above method, in step 4, the multiple resuspensions are specifically:

[0017] Step 401: Resuspend in lysis buffer, gently rotate at 4°C for 5 minutes, place on a magnetic rack, and discard the supernatant;

[0018] Step 402: Resuspend in buffer 1, gently rotate at 4°C for 5 minutes, place on a magnetic rack, and discard the supernatant;

[0019] Step 403: Resuspend in buffer 2, gently rotate at 4°C for 5 minutes, place on a magnetic rack, and discard the supernatant;

[0020] Step 404: Resuspend in buffer 3, gently rotate at 4°C for 5 minutes, place on a magnetic rack, and discard the supernatant;

[0021] Step 405: eluting with an eluent;

[0022] The buffer 1 contains 20 mM Tris-HCl, 500 mM LiCl, 0.1% LiDS, 1 mM EDTA, and 5 mM DTT;

[0023] The buffer 2 contains 20 mM Tris-HCl, 500 mM LiCl, 1 mM EDTA, and 5 mM DTT;

[0024] The buffer 3 contains 20 mM Tris-HCl, 200 mM LiCl, 1 mM EDTA, and 5 mM DTT;

[0025] The eluent contains 20 mM Tris-HCl and 1 mM EDTA;

[0026] The lysis buffer contains 20 mM Tris, 500 mM LiCl, 0.5% lithium dodecylsulfate, 1 mM EDTA, and 5 mM DTT.

[0027] In the above method, the lysis buffer is divided into three portions, and steps 401 to 405 are repeated for each portion of the lysis buffer. The capture operation of RNA-binding proteins is performed on the three portions of the lysis buffer, and then the RNA-binding proteins captured from the three portions of the lysis buffer are combined.

[0028] In the above method, step 1 is specifically as follows:

[0029] Step 11: After separating the muscle tissue or subcutaneous adipose tissue, place it in PBS containing penicillin-streptomycin and wash to remove the fascia.

[0030] Step 12: Cut the muscle tissue or subcutaneous adipose tissue into minced meat, add cell separation buffer, and digest it in a shaker at 37°C for 1 h. During this period, pipette every 10 min until the tissue pieces completely disappear and become milky, and the digestion solution is obtained. The composition of the cell separation buffer is: 0.1 wt% type I collagenase, 120 mM HEPES, 100 mM NaCl, 50 mM KCl, 1 mM glucose, 1 mM CaCl2, 1.5% BSA, and finally adjust the pH value to 7.4;

[0031] Step 13: Neutralize the digestion solution with complete medium, filter and centrifuge, and collect the supernatant centrifugate;

[0032] Step 14: Dilute and wash the supernatant with DMEM medium, centrifuge, take the supernatant centrifugate, and add it to a clean 10 cm culture dish, which is the mature intramuscular adipocytes or subcutaneous adipocytes.

[0033] Step 14 is more specific as follows: Add the supernatant centrifugate collected in Step 13 to 10 ml of pre-cooled PBS to wash the cells, then centrifuge at 1000 g for 10 min, carefully aspirate 0.5 mL of the supernatant centrifugate, and finally add it to a clean 10-cm culture dish, ensuring that the monolayer of adipocytes covers the culture dish, and avoiding excessive thickness that may cause insufficient irradiation of the bottom cells.

[0034] In the above method, Step 2 is specifically as follows:

[0035] Immediately open the lid of the culture dish and place it on ice 10 - 30 cm away from the ultraviolet light source, and irradiate with UV light of 0.15 J / cm 2 for 1 minute, and the wavelength of the UV light is 254 nm.

[0036] In the above method, Step 3 is specifically as follows:

[0037] Collect the cells in the culture dish, centrifuge and remove the supernatant, then add lysis buffer and pipette up and down; then homogenize multiple times and incubate at 4 °C for 10 minutes;

[0038] The lysis buffer contains 20 mM Tris, 500 mM LiCl, 0.5% lithium dodecylsulfate, 1 mM EDTA, 5 mM DTT. Preferably, it also contains protease inhibitors and RNase inhibitors.

[0039] In the above method, Step 5 is specifically as follows:

[0040] Step 51: Take 20 μl of the eluate obtained in Step 4, add 5 μl of 5×proteinase K buffer and 1 μg of proteinase K buffer, and incubate at 50 °C for 1 hour;

[0041] Step 52: Add trizol reagent to the product of Step 51 to extract RNA, and use the qPCR method to detect the levels of actb and 18s to prove the successful enrichment of RNA;

[0042] Step 53: Add 500 μl of 10×ribonuclease buffer, 200 U of RNase T1 and RNase A to the remaining eluate of the sample collected in Step 4, and incubate at 37 °C for 1 hour;

[0043] Step 54: Transfer the eluate after incubation to an ultrafiltration centrifuge tube retention device to retain products with a molecular weight cutoff of 3 kD or more. Fill the ultrafiltration centrifuge tube retention device to the brim with buffer 4, and then centrifuge at 4°C. Discard the filtrate, fill the ultrafiltration centrifuge tube retention device to the brim with buffer 4 again, centrifuge at 4°C, and then recover the sample from the ultrafiltration centrifuge tube retention device.

[0044] Step 55: Determine the protein concentration of the sample obtained in step 54, perform silver staining, and perform western blot detection to prove that the RNA binding protein is successfully enriched and that the sample obtained in step 54 is an RNA binding protein.

[0045] In the above method, if the sample is muscle tissue, the RNA-binding proteins isolated in step 5 are WDR82 protein, NFKBIE protein, AFTPH protein, ARL13A protein, TENM1 protein, PGM2 protein, RIMS2 protein, SH2D3C protein, ZNF821 protein, MBD4 protein, MUS81 protein, MYBPC2 protein, ACACA protein, MPP7 protein, JMJD7 protein, FGF12 protein, SLC4A4 protein, DNAH5 protein, MAPKAP1 protein, and IDH3B protein;

[0046] If the sample is subcutaneous adipose tissue, the RNA-binding proteins isolated in step 5 are PCDH1 protein, BAIAP2 protein, RABEP2 protein, DPYSL4 protein, TUBGCP5 protein, FBRS protein, SPTY2D1 protein, ACTB protein, SIK2 protein, VOPP1 protein, GPR143 protein, MLXIPL protein, OBI1 protein, LSS protein, RGS4 protein, LOC100510917 protein, SH3GL3 protein, TPM4 protein, ZNF335 protein, and INO80 protein.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention uses UV cross-linking and curing method to treat intramuscular adipocytes or subcutaneous adipocytes, and then uses magnetic beads to capture RNA binding proteins, successfully obtaining RNA binding proteins, filling the gap in the industry where there is no mature method for extracting RNA binding proteins from intramuscular adipocytes or subcutaneous adipocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The photos of UV cross-linked (CL) and non-cross-linked (NoCL) halos;

[0050] Figure 2A Graph showing the test results of the sample of the present invention;

[0051] Figure 2B This is the test result diagram of the non-cross-linked sample;

[0052] Figure 3 This is a silver staining result diagram of the sample of the present invention;

[0053] Figure 4 It is a western blot detection result diagram of the sample of the present invention;

[0054] Figure 5 This is a diagram showing the overlap between the RNA-binding proteins in pig intramuscular adipocytes of Example 1 and the RNA-binding proteins of human and mouse origin. DETAILED DESCRIPTION

[0055] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0056] Source and description of reagents

[0057] Penicillin-streptomycin: Thermo Fisher Scientific, 15140122;

[0058] PBS: Thermo Fisher Scientific, C10010;

[0059] Collagenase type Ⅰ: Thermo Fisher Scientific, 17018029;

[0060] DMEM medium: Thermo Fisher Scientific, C11995500;

[0061] Fetal bovine serum: Thermo Fisher Scientific, 10099141;

[0062] NP-40: Thermo Fisher Scientific, 85125;

[0063] Proteinase K: Thermo Fisher Scientific, EO0491;

[0064] Trizol reagent: Thermo Fisher Scientific, 15596018CN;

[0065] RNase T1: Sigma-Aldrich, R1003;

[0066] RNase A: Sigma-Aldrich, R4642.

[0067] Example 1

[0068] A method for identifying RNA-binding proteins in porcine intramuscular adipocytes, comprising the following steps:

[0069] Step 1: Isolation of porcine primary mature adipocytes

[0070] In this step, a mild cell isolation buffer is used, which specifically includes the following sub-steps:

[0071] Step 11: After isolating the longissimus dorsi muscle of piglets, place it in PBS containing penicillin-streptomycin and wash it, and use a sharp scissors to remove the surface fascia completely.

[0072] Step 12: After cutting the muscle into minced meat with a sharp scissors, add 20 ml of cell isolation buffer, and oscillate and digest it in a shaker at 37 °C for 1 h. During this period, pipette once every 10 min until the tissue block completely disappears into a milky state and ends to obtain a digestive solution. The composition of the cell isolation buffer is: 0.1 wt% type I collagenase, 120 mM HEPES, 100 mM NaCl, 50 mM KCl, 1 mM glucose, 1 mM CaCl2, 1.5% BSA, and finally adjust the pH value to 7.4;

[0073] Step 13: Neutralize the digestive solution with an equal volume of complete medium, filter it through a 200-mesh sieve, and centrifuge it at a centrifugal force of 800 g for 10 min. Collect 4 ml of the supernatant centrifugate into a new centrifuge tube.

[0074] Step 14: Add the supernatant centrifugate containing mature intramuscular adipocytes collected in Step 13 to 10 ml of pre-cooled PBS to wash the cells, then centrifuge at 1000 g for 10 min, carefully aspirate 0.5 mL of the supernatant centrifugate, and finally add it to a clean 10-cm culture dish to ensure that the intramuscular adipocytes form a monolayer covering the culture dish, avoiding excessive thickness resulting in insufficient irradiation of the bottom cells.

[0075] Since UV irradiation preferably has a good effect on monolayer cells, for adherent cells, only by completely removing the medium in the culture dish can the purpose of monolayer cells be achieved. Freshly isolated and mature adipocytes cannot adhere to the wall and float on the upper layer of the liquid after centrifugation. Therefore, 0.5 ml of the supernatant is selected and added to a 10-cm culture dish. The volume of this liquid just covers the 10-cm culture dish, which can basically ensure that the mature adipocytes are in a monolayer state.

[0076] This step is relatively crucial and is the guarantee for achieving effective subsequent UV cross-linking. During the preliminary exploratory experiment process, we used 1 ml of the supernatant. After UV irradiation, it was found that it could not effectively cross-link the cells completely, making the subsequent experiment impossible to proceed.

[0077] Step 2: Solution Preparation - UV Crosslinking - Cell Lysis - Enrichment and Elution of RNA-Binding Proteins - Proteomic Analysis Step 21: Solution Preparation:

[0078] Lysis buffer: 20 mM Tris (pH 7.5), 500 mM LiCl, 0.5% lithium dodecyl sulfate (LiDS), 1 mM EDTA, 5 mM DTT. Autoclave the buffer before adding LiDS and DTT. After cooling, complete the buffer by adding LiDS and DTT, and then filter.

[0079] Buffer 1: 20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 0.1% LiDS (wt / vol), 1 mM EDTA, 5 mM DTT.

[0080] Buffer 2: 20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 5 mM DTT.

[0081] Buffer 3: 20 mM Tris-HCl (pH 7.5), 200 mM LiCl, 1 mM EDTA, 5 mM DTT.

[0082] Elution buffer: 20 mM Tris-HCl (pH 7.5), 1 mM EDTA.

[0083] Buffer 4: 10 mM Tris-HCl (pH 7.5), 50 mM NaCl.

[0084] RNase buffer (ribonuclease buffer), 10×: 100 mM Tris-HCl (pH 7.5), 1.5 M NaCl, 0.5% (vol / vol) NP-40, 5 mM DTT.

[0085] Proteinase K buffer, 5×: 50 mM Tris-HCl (pH 7.5), 750 mM NaCl, 1% (wt / vol) sodium dodecyl sulfate (SDS), 50 mM EDTA, 2.5 mM DTT, 25 mM CaCl2.

[0086] Step 22: Wash the cells obtained in Step 14 twice with PBS;

[0087] Step 23: UV cross-linking: Remove PBS from the solution in Step 22, place the cells in a culture dish, open the lid of the culture dish and place it on ice at a distance of 10 - 15 - 30 cm from the UV, irradiate with 0.15 J / cm 2 of UV (wavelength 254 nm) for 1 minute, add cold PBS and place on ice, using the uncross-linked sample (noCL) as a control.

[0088] Step 24: Scrape the cells irradiated in Step 23 with a cell scraper, centrifuge at a centrifugal force of 400 g for 3 minutes, and discard the supernatant.

[0089] Step 25: Cell lysis, add lysis buffer to the cells in Step 23 and pipette up and down.

[0090] Step 26: Homogenize the sample obtained in Step 25 three times with a syringe equipped with a narrow needle (diameter 0.4 mm), incubate at 4 °C for 10 minutes, and collect 2% INPUT.

[0091] Step 27: Take an appropriate amount of oligo(dT) magnetic beads and wash them 3 times with lysis buffer;

[0092] Step 28: Mix the magnetic beads obtained in Step 27 with the sample obtained in Step 26 and rotate at 4 °C for 1 - 2 hours;

[0093] Step 29: Place the sample obtained in Step 28 on a magnetic stand at 4 °C, collect the supernatant into a new centrifuge tube for the other two magnetic bead captures;

[0094] Step 210: Resuspend the magnetic beads with lysis buffer, with the volume ratio of magnetic beads to lysis buffer being, gently rotate at 4 °C for 5 minutes, place on a magnetic stand, and discard the supernatant;

[0095] Step 211: Resuspend with buffer 1, gently rotate at 4 °C for 5 minutes, place on a magnetic stand, and discard the supernatant, repeat once. Refer to Figure 1 , at this time, a magnetic bead halo can be observed, proving that the mRNA - protein complex has been enriched;

[0096] Step 212: Resuspend with buffer 2, gently rotate at 4 °C for 5 minutes, place on a magnetic stand, and discard the supernatant, repeat once;

[0097] Step 213: Resuspend with buffer 3, gently rotate at 4 °C for 5 minutes, place on a magnetic stand, and discard the supernatant, repeat once;

[0098] Step 214: Elute the magnetic beads obtained in Step 213 with 500 μl of eluent at 55 °C for 3 minutes, and measure the RNA concentration with a UV spectrophotometer.

[0099] Step 215: For the supernatant obtained in Step 29, repeat Steps 210 - 214 twice using magnetic beads;

[0100] Step 216: Combine the samples eluted above three times, for a total of 1.5 ml.

[0101] Step 217: Take 20 μl of the sample obtained in Step 216, add 5 μl of 5×proteinase K buffer, 1 μg of proteinase K, and incubate at 50 °C for 1 hour;

[0102] Step 218: Add trizol reagent to extract RNA, and detect the levels of actb and 18s by qPCR (the detection results are for reference Figure 2A and Figure 2B , Figure 2A are the detection results of the samples of the present invention, Figure 2B are the detection results of the uncrosslinked samples), and it can be proved by the figure that the RNA enrichment of the present invention is successful;

[0103] Step 219: Add 500 μl of 10×ribonuclease buffer and approximately 200 U of RNase T1 and RNase A to the remaining combined eluate (4480 μl) in Step 216. Incubate the mixture at 37 °C for 1 hour. Transfer the ribonuclease-treated eluate to an ultrafiltration centrifugal tube retention device Amicon Ultra 10 (retention molecular weight 3 kDa, a 50 ml device is used for large-scale experiments, and a 2 ml device is used for small-scale experiments). Fill the ultrafiltration centrifugal tube retention device with buffer 4 until full, and then centrifuge at 4000 g at 4 °C for 45 minutes.

[0104] Step 220: Discard the filtrate, and add the sample to the ultrafiltration centrifugal tube retention device again with buffer 4 until full. Centrifuge at 4000 g at 4 °C for 45 minutes.

[0105] Step 221: Recover the sample (about 200 μl) from the ultrafiltration centrifugal tube retention device.

[0106] Step 222. Detection:

[0107] The detection items include:

[0108] Measuring protein concentration, silver staining, and western blot detection;

[0109] Refer to Figure 3 , Figure 3 is the silver staining result, and it can be proved by Figure 3 that the protein enrichment is successful;

[0110] Refer to Figure 4 ,Figure 4 This is the result of western blot detection. The known RNA-binding protein YBX1 was used as the positive control, and the non-RNA-binding proteins ACTB and tubulin were used as the negative controls. Figure 4 It was demonstrated that the enriched protein is an RNA-binding protein.

[0111] Through further analysis, 405 RNA-binding proteins were screened out in this example. Referring to Figure 5 , among which 151 proteins are RNA-binding proteins different from those of mice and humans. Table 1 below shows some of these 151 RNA-binding proteins.

[0112] Example 2

[0113] A method for identifying RNA-binding proteins in adipocytes of subcutaneous adipose tissue, which comprises the following steps:

[0114] Step 1: Isolation of porcine primary mature adipocytes

[0115] In this step, a mild cell isolation buffer was used, which specifically includes the following sub-steps:

[0116] Step 11: After isolating the subcutaneous adipose tissue of piglets, it was placed in PBS containing penicillin-streptomycin and washed.

[0117] Step 12: After cutting the subcutaneous adipose tissue into pieces with a sharp scissors, 20 ml of the cell isolation buffer described in Example 1 was added, and it was digested by shaking in a 37 °C shaker for 1 h. During this period, pipetting was performed every 10 min until the tissue pieces completely disappeared into a milky state, and the digestion solution was obtained.

[0118] Step 13: Neutralize the digestion solution with an equal volume of complete medium, filter it through a 200-mesh sieve, and centrifuge it at a centrifugal force of 800 g for 10 min. Collect 4 ml of the supernatant centrifugate into a new centrifuge tube.

[0119] Step 14: Add the supernatant centrifugate collected in Step 13 to 10 ml of pre-cooled PBS, wash the cells, then centrifuge at 1000 g for 10 min, carefully aspirate 0.5 mL of the supernatant centrifugate, and finally add it to a clean 10-cm culture dish to ensure that the cell monolayer covers the culture dish, avoiding excessive thickness resulting in insufficient irradiation of the bottom cells.

[0120] Step 2: Solution preparation - ultraviolet cross-linking - cell lysis - enrichment and elution of RNA-binding proteins - proteomic detection and analysis;

[0121] This step is exactly the same as that in Example 1;

[0122] Table 1 List of names of some RNA-binding proteins identified in this example

[0123]

[0124]

[0125] Comparative Example 1

[0126] The method is substantially the same as Example 1, except that the elution buffer is prepared with 20 mM Tris-HCl (pH 7.5) and does not contain EDTA.

[0127] In the experiment, we found that whether the eluent contained EDTA had a certain influence on the results, which limited the number of RNA-binding proteins screened. In this comparative example 1, the number of RNA-binding proteins screened was 213, which was lower than that in Example 1.

[0128] Comparative Example 2

[0129] The method is substantially the same as Example 1, except that the buffer 2 comprises 20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 5 mM DTT, and 0.02% (v / v) NP40 (detergent).

[0130] Buffer 3: 20 mM Tris-HCl (pH 7.5), 200 mM LiCl, 1 mM EDTA, 5 mM DTT, 0.02% (v / v) NP40 (detergent).

[0131] The purpose of NP40 is to remove the interference of impurity ions. In the preliminary experiments of this project, we attempted to use NP40 to remove the possible influence of impurities. After experiments, we found that it was unable to screen out the number of RNA-binding proteins shown in Example 1. This comparative example 2 was only able to screen out 161 RNA-binding proteins.

[0132] In summary, the core innovation of the present invention lies in:

[0133] 1. The present invention is the first to achieve the exploration of the separation of RNA-binding proteins in adipocytes;

[0134] 2. Compared with traditional tissue cells that are easy to adhere to the wall, the adipocytes of the present invention are difficult to adhere to the wall. In order to facilitate the subsequent magnetic bead enrichment operation, the present invention optimizes the operation in step 14 so that the adipocytes appear as a single layer of cells in the culture medium, which is conducive to UV irradiation.

[0135] 3. The present invention has high separation efficiency for RNA-binding proteins of adipocytes, realizes the definition of RNA-binding proteins of existing proteins and completely distinguishes them from those of human and mouse genera, which has positive significance for subsequent work in drug targets, genetic breeding, etc.

Claims

1. A method for identifying RNA-binding proteins in adipocytes, characterized in that, It includes the following steps: Step 1: Take muscle tissue or subcutaneous adipose tissue, mince and digest it, and collect mature intramuscular adipocytes or subcutaneous adipocytes; Step 2: Wash the intramuscular adipocytes or subcutaneous adipocytes and then perform cross-linking treatment by ultraviolet irradiation; Step 3: Collect the cross-linked intramuscular adipocytes or subcutaneous adipocytes, lyse them under the action of cell lysate to obtain a lysate; Step 4: Add magnetic beads to the lysate, resuspend it multiple times and wash it to obtain an eluate; Step 5: Separate the RNA-binding protein from the magnetic beads and incubate it.

2. The method according to claim 1, wherein In step 4, the multiple resuspensions are specifically as follows: Step 401: Resuspend with lysis buffer lysate, gently rotate at 4°C for 5 minutes, place on a magnetic stand, and discard the supernatant; Step 402: Resuspend with buffer 1, gently rotate at 4°C for 5 minutes, place on a magnetic stand, and discard the supernatant; Step 403: Resuspend with buffer 2, gently rotate at 4°C for 5 minutes, place on a magnetic stand, and discard the supernatant; Step 404: Resuspend with buffer 3, gently rotate at 4°C for 5 minutes, place on a magnetic stand, and discard the supernatant; Step 405: Elute with eluate; The buffer 1 contains 20 mM Tris-HCl, 500 mM LiCl, 0.1% LiDS, 1 mM EDTA, 5 mM DTT; The buffer 2 contains 20 mM Tris-HCl, 500 mM LiCl, 1 mM EDTA, 5 mM DTT; The buffer 3 contains 20 mM Tris-HCl, 200 mM LiCl, 1 mM EDTA, 5 mM DTT; The eluate contains 20 mM Tris-HCl, 1 mM EDTA; The lysis buffer lysate contains 20 mM Tris, 500 mM LiCl, 0.5% lithium dodecylsulfate, 1 mM EDTA, 5 mM DTT.

3. The method according to claim 2, wherein Divide the lysate into three portions, repeat steps 401 to 405 for each portion of the lysate, perform the capture operation of RNA-binding protein on the three portions of the lysate, and then combine the captured RNA-binding proteins from the three portions of the lysate.

4. The method according to claim 1, wherein Step 1 is specifically as follows: Step 11: After separating the muscle tissue or subcutaneous adipose tissue, place it in PBS containing penicillin-streptomycin and wash it to remove fascia. Step 12: Cut the muscle tissue or subcutaneous adipose tissue into minced meat, add cell separation buffer, and oscillate and digest it in a 37°C shaker for 1 h. During this period, pipette every 10 min until the tissue pieces completely disappear and become milky, and end to obtain a digestive solution; the composition of the cell separation buffer is: 0.1 wt% type I collagenase, 120 mM HEPES, 100 mM NaCl, 50 mM KCL, 1 mM glucose, 1 mM CaCl2, 1.5% BSA, and finally adjust the pH value to 7.4; Step 13: Neutralize the digestive solution with complete medium, filter and centrifuge, and collect the supernatant centrifugate; Step 14: Add DMEM medium to the supernatant for dilution, washing, and centrifugation. Take the supernatant centrifugate and add it to a clean 10-cm culture dish, which is the mature intramuscular adipocytes or subcutaneous adipocytes.

5. The method according to claim 1, characterized in that, The specific steps of step 2 are as follows: Immediately open the lid of the petri dish and place it on ice at a distance of 10 - 30 cm from the ultraviolet light source, and irradiate it with UV light of 0.15 J / cm 2 for 1 minute. The wavelength of the UV light is 254 nm.

6. The method according to claim 5, characterized in that, The specific steps of step 3 are as follows: Immediately add lysis buffer, pipette up and down; then homogenize multiple times and incubate at 4°C for 10 minutes; the lysis buffer contains 20 mM Tris, 500 mM LiCl, 0.5% lithium dodecyl sulfate, 1 mM EDTA, 5 mM DTT. Preferably, it also contains protease inhibitors and RNase inhibitors.

7. The method according to claim 1, characterized in that The specific steps of step 5 are as follows: Step 51: Take 20 μl of the eluate obtained in step 4, add 5 μl of 5×proteinase K buffer and 1 μg of proteinase K buffer, and incubate at 50°C for 1 hour. Step 52: Add trizol reagent to the product of step 51 to extract RNA, and use the qPCR method to detect the levels of actb and 18s to prove successful RNA enrichment. Step 53: Add 500 μl of 10×ribonuclease buffer, 200 U of RNase T1, and RNase A to the remaining eluate of the sample collected in step 4, and incubate at 37°C for 1 hour. Step 54: Transfer the incubated eluate to an ultrafiltration centrifugal tube retention device to retain products with a molecular weight above 3 KD. Fill the ultrafiltration centrifugal tube retention device with buffer 4, then centrifuge at 4°C; discard the filtrate, add the sample to the ultrafiltration centrifugal tube retention device again with buffer 4 until full, centrifuge at 4°C, and then recover the sample from the ultrafiltration centrifugal tube retention device. Step 55: Measure the protein concentration of the sample obtained in step 54, perform silver staining, and perform western blot detection to prove successful enrichment of RNA-binding protein and prove that the sample obtained in step 54 is RNA-binding protein.

8. The method according to claim 1, characterized in that, The specific steps of step 14 are as follows: Add the supernatant centrifugate collected in step 13 to 10 ml of pre-cooled PBS to wash the cells, then centrifuge at 1000 g for 10 min, carefully aspirate 0.5 mL of the supernatant centrifugate, and finally add it to a clean 10-cm culture dish to ensure that the adipocyte monolayer covers the culture dish, avoiding excessive thickness that may cause insufficient irradiation of the underlying cells.

9. The method according to claim 1, characterized in that, If the sample is muscle tissue, the RNA-binding proteins isolated in step 5 are WDR82 protein, NFKBIE protein, AFTPH protein, ARL13A protein, TENM1 protein, PGM2 protein, RIMS2 protein, SH2D3C protein, ZNF821 protein, MBD4 protein, MUS81 protein, MYBPC2 protein, ACACA protein, MPP7 protein, JMJD7 protein, FGF12 protein, SLC4A4 protein, DNAH5 protein, MAPKAP1 protein, IDH3B protein; If the sample is subcutaneous adipose tissue, the RNA-binding proteins isolated in the said step 5 are PCDH1 protein, BAIAP2 protein, RABEP2 protein, DPYSL4 protein, TUBGCP5 protein, FBRS protein, SPTY2D1 protein, ACTB protein, SIK2 protein, VOPP1 protein, GPR143 protein, MLXIPL protein, OBI1 protein, LSS protein, RGS4 protein, LOC100510917 protein, SH3GL3 protein, TPM4 protein, ZNF335 protein, and INO80 protein.

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