RNA co-immunoprecipitation kit for specific binding prion protein in muscle cells and RNA extraction method

Abstract

The invention discloses an RNA co-immunoprecipitation kit for specific binding of prion protein in muscle cells and an RNA extraction method, and relates to the technical field of bioengineering. According to the present invention, the specific co-precipitation kit preparation and the specific extraction steps comprise cell lysis, cell lysis solution-antibody-magnetic bead incubation, protein digestion and RNA extraction, such that the high purity and the sufficient amount of the enriched RNA are successfully ensured so as to completely meet the strict requirements of the subsequent RNA-seq sequencing. The method overcomes the inherent defects of the existing RIP technology in the research of non-classical RNA binding proteins, realizes the optimization of the whole process from high-specificity enrichment to high-quality sequencing sample preparation through the integrated innovation of methodology and the kit, has the outstanding advantages of strong specificity, high sensitivity, good repeatability, simple operation and the like, and has a wide application prospect in the research of non-classical RNA binding proteins. And remarkable technical progress is achieved, and a positive application effect is generated.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an RNA immunoprecipitation kit and RNA extraction method for specifically binding prion proteins in muscle cells. Background Technology

[0002] RNA immunoprecipitation (RIP) is a key technique used under natural physiological conditions to study the binding of endogenous RNA to specific proteins within cells. This technique utilizes specific antibodies against the target protein to immunoprecipitate the bound RNA-protein complex, followed by the separation and purification of the RNA components. This is then combined with high-throughput sequencing (such as RIP-seq) or molecular biology analysis methods to identify, quantify, and functionally analyze the bound RNA. As a powerful tool for revealing the dynamic processes of post-transcriptional regulatory networks (such as mRNA stability regulation, translation efficiency regulation, and non-coding RNA functional mechanisms), RIP technology is particularly widely used to discover regulatory targets of microRNAs (miRNAs), recognize substrates of RNA-binding proteins (RBPs), and explore the core role of RNA in cellular function and disease development.

[0003] Although RIP technology has been widely used in the study of various RNA-binding proteins, it faces significant bottlenecks and challenges when studying non-classical RNA-binding proteins, especially those lacking typical RNA-binding domains (such as RRM, KH, DSR, etc.). This constitutes a major limitation of existing technology: firstly, the "non-classical" nature of the target protein creates a technical blind spot. Traditional RIP technology and its data analysis strategies largely rely on the study of known or predicted classical RNA-binding proteins. For proteins like prions (PrP... C Proteins that lack a universally recognized classic RNA-binding domain but can functionally interact with nucleic acids through atypical regions are often under-researched or subject to technical bias in existing research systems. C As a key membrane protein highly expressed in the nervous system and undergoing conformational changes in prion diseases, PrPC, while lacking a classic DNA / RNA binding domain, possesses multiple polypeptide chain regions that potentially mediate nucleic acid interactions, as revealed by structural biology and biochemical studies. These regions include two lysine-rich clusters (residues 23-31 and 101-110), five octapeptide repeat sequences, and a C-terminal domain. These regions constitute at least eight potential nucleic acid binding or cross-linking sites. However, due to the lack of a classic binding domain, PrPC's interaction with RNA has been easily overlooked in previous studies, and the types of interacting RNAs, binding specificity, affinity, and biological significance remain largely unclear. Furthermore, the low abundance and high complexity of interacting RNAs present challenges for enrichment and identification. For proteins like PrPC... CProteins that may engage in transient, dynamic, or low-affinity RNA interactions may have low intracellular RNA abundance and high background noise. Conventional RIP (Rapid Injection Propagation) procedures may be insufficient in terms of RNA enrichment efficiency, purity, and sensitivity for subsequent analyses to effectively capture and identify these low-abundance or weakly bound RNA molecules. This means that even with RIP experiments, it may be impossible to obtain a sufficient quantity and high purity of RNA for downstream high-throughput sequencing (such as RNA-seq), making it difficult to comprehensively and unbiasedly reveal their interacting RNA profile. Furthermore, functional association analysis also presents challenges; even if proteins with PrP are successfully identified... C How to distinguish between specific binding and non-specific adsorption of bound RNA, and further elucidate these RNA-PrP bindings. C The specific biological functions of interactions in muscle cells (or other cell types) (such as whether they affect RNA stability, localization, translation, or participate in cellular stress responses and signal transduction) represent another significant bottleneck in current technologies. The lack of effective functional validation strategies means that interaction data will remain merely descriptive of correlations, making it difficult to translate into meaningful analyses of PrP. C Mechanistic understanding of biological functions or pathological mechanisms. Summary of the Invention

[0004] This invention specifically focuses on the application and optimization of RNA immunoprecipitation (RIP) technology, aiming to solve the problem of efficient and specific enrichment of PrP in muscle cells. C This invention addresses the critical issue of RNA-protein interactions. By precisely optimizing experimental conditions (crosslinking strategy, lysis buffer composition, Wash buffer composition, and proteinase K buffer composition), it successfully ensured the high purity and sufficient quantity of enriched RNA, fully meeting the stringent requirements of subsequent RNA-seq sequencing. This technological achievement paves the way for overcoming the challenges posed by the "non-classical" RNA-binding protein PrP. C This provides a reliable methodological basis for solving the challenge of identifying interacting RNAs, enabling systematic revelation of PrP. C This study has made it possible to explore the potential RNA interaction network in muscle cells and its regulatory role in cellular physiological or pathological processes. This research holds promise for expanding our understanding of PrP... C The understanding of non-classical functions also provides a reference for the technical path and analytical paradigm for studying the interaction between other proteins that lack typical RNA-binding domains and nucleic acids.

[0005] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides an RNA immunoprecipitation kit for specifically binding prion proteins in muscle cells, comprising RIP lysis buffer, Wash buffer, proteinase K buffer, and pre-use additives. The RIP lysis buffer comprises: 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol. The Wash buffer solution comprises: 25 mM Tris-HCl pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol; The proteinase K buffer comprises: 10 μL 1 M Tris-HCl pH 7.4, 10 μL 5 M NaCl, 10 μL 0.5 M EDTA, 50 μL 10% SDS, 60 μL 20 mg / mL proteinase K solution, 10 μL 40 U / μL RNase inhibitor, 2 μL 0.5 M DTT, and 848 μL DEPC water; The pre-use additives are added to RIP lysis buffer and Wash buffer before use. The pre-use additives include RNase inhibitors, DTT and protease inhibitors, and the concentrations of the three after addition are RNase inhibitor 400 U / ml, DTT 1mM and protease inhibitor 1×, respectively.

[0006] Furthermore, the RNase inhibitor, DTT, and protease inhibitor are each packaged separately.

[0007] Furthermore, it also includes pre-packaged Protein A / G magnetic bead suspensions.

[0008] Secondly, based on the kit provided in the first aspect, this invention discloses a method for extracting RNA that specifically binds to prion proteins in muscle cells, comprising the following steps: S10: Differentiation induction treatment: muscle cells were placed in a cell culture dish, DMEM culture medium was added, and then 1% formaldehyde solution prepared with PBS was added. After standing at room temperature for 10 min, 1.25 M glycine solution prepared with PBS was added dropwise and then stood at room temperature for 5 min. S20: Wash twice with pre-chilled PBS and then add pre-chilled PBS. Scrape cells with a cell scraper under ice and centrifuge to collect the cell pellet. Add RIP lysis buffer with pre-use additive to the cell pellet and lyse on ice for 20 minutes, gently pipetting every 10 minutes during the lysis. After lysis, centrifuge to collect the supernatant as the cell lysis buffer. S30: Take the cell lysate and place it in an EP tube, then add anti-PrP. C Monoclonal antibody 8H4 was incubated at 4°C for 5 hours by rotation. S40: Take a new EP tube, add Protein A / G magnetic beads to the EP tube, wash twice with PBS and discard the supernatant, then wash twice with Wash buffer and discard the supernatant, and finally wash once with Wash buffer containing the pre-use additive and discard the supernatant. S50: Add the antibody-lysis buffer mixture incubated in S30 to the treated Protein A / G magnetic beads in S40 and incubate at 4°C for 3 hours by rotation. After incubation, centrifuge and discard the supernatant, and wash the magnetic beads twice with Wash buffer containing pre-use additives, centrifuge and discard the supernatant. S60: Add proteinase K buffer to the magnetic beads and incubate at 55°C for 5 minutes to digest the protein. After incubation, immediately place on ice to cool. S70: Add Trizol to the digested sample, shake vigorously for 15 seconds, let stand at room temperature for 10 minutes, then add chloroform, shake vigorously again for 15 seconds, let stand at room temperature for 15 minutes; then centrifuge to collect the supernatant into a new EP tube, add an equal volume of isopropanol and shake for 15 seconds, then add 1 μL of Glycogen blue, let stand on ice for 10 minutes, then incubate overnight at -80℃ to precipitate RNA, centrifuge the next day and discard the supernatant; S80: Wash the RNA precipitate twice with 75% ethanol under ice bath; after air drying at room temperature for 10 minutes, dissolve the RNA in DEPC water, add RNase inhibitor, determine the concentration, and then freeze in liquid nitrogen for later use.

[0009] More preferably, in step S10, the muscle cells are washed twice with pre-cooled PBS before being placed in the cell culture dish.

[0010] In summary, compared with the prior art, the present invention has the following advantages and benefits: 1. This invention overcomes the research bottleneck of "non-classical" RNA-binding proteins through specific kits and procedures, achieving highly specific enrichment. Existing RIP technology and its conventional reagent systems are mainly optimized for proteins with typical RNA-binding domains, but for proteins like PrP... C Proteins lacking classic binding domains and primarily interacting with nucleic acids through atypical regions such as lysine-rich clusters and octapeptide repeat sequences suffer from technical drawbacks including poor specificity, high background noise, and weak effective signals. This invention, through a unique antibody selection strategy and optimized cell lysis buffer and wash buffer formulations (especially ionic strength and detergent system), significantly enhances the immunoprecipitation process for PrP. C The targeted capture capability of the RNA complex was enhanced while minimizing the co-precipitation of non-specific nucleic acids. Thus, for the first time, the targeting of PrP-RNA complexes under the physiological environment of muscle cells was achieved. CThe efficient enrichment of RNAs that exhibit specific, potentially low-affinity, or transient interactions addresses a blind spot in existing technologies for the study of such target proteins.

[0011] 2. This invention significantly improves the yield and purity of interacting RNA, ensuring direct compatibility with downstream high-throughput sequencing. (Regarding PrP) C Interacting RNAs may be low in abundance and easily degraded. Existing RIP methods often face problems such as insufficient eluted RNA and severe interference from impurities (e.g., non-bound RNA, protein residues, reagent impurities), leading to the need for additional RNA amplification or purification steps. This not only introduces bias but also increases operational complexity and sample loss. The integrated kit and standardized operating procedure provided by this invention, through synergistic optimization of cross-linking conditions, RNase inhibitors, complex dissociation, and RNA purification steps, ensures high efficiency and stability throughout the entire process from cell lysis to RNA recovery. The final RNA product obtained is of high purity, good integrity, and sufficient yield, fully meeting the library preparation requirements for direct next-generation sequencing (RNA-seq). No intermediate amplification step is required, maximizing the preservation of the original interacting RNA profile and enabling unbiased and comprehensive analysis of PrP. C The RNA binding genome (RBPome) laid a reliable technological foundation.

[0012] 3. This invention establishes a standardized operating system, improving the reproducibility and ease of application of the experiment. Existing research on PrP C The lack of standardized protocols for RNA interaction methods, coupled with significant differences in conditions between laboratories, makes it difficult to compare and replicate results. This invention transforms an optimized method into a ready-to-use detection kit, containing all necessary pre-optimized reagents and detailed operating instructions. This significantly reduces the technical barrier and operational complexity, enabling researchers without extensive RIP experience to stably and reproducibly perform PrP in muscle cell models. C Enrichment and preparation of specific RNA-intercrops will facilitate the promotion and application of this technology and the comparability of data between different studies.

[0013] 4. This invention provides an in-depth analysis of PrP C It provides crucial tools for understanding non-classical functions and pathological mechanisms in muscle cells. Traditionally, PrP... C Functional studies have focused on its role in the central nervous system. This invention provides, for the first time, a specific target for PrP in muscle cells. C A reliable tool for systematic research on RNA interactions. The high-quality RNA obtained using this invention, which can be directly used for RNA-seq, enables precise identification of RNAs interacting with PrP in muscle cells. CSpecifically binding to mRNAs and non-coding RNAs (such as miRNAs and lncRNAs), and then through bioinformatics analysis and functional experiments, the PrP pathway can be revealed. C Potential novel functions of PrP in posttranscriptional regulation, stress response, metabolism, or signal transduction in muscle cells, and its possible association with muscle-related diseases. This contributes to a comprehensive understanding of PrP. C This has opened up new research directions for the biological significance of the disease and the development of novel diagnostic and treatment strategies for related diseases.

[0014] 5. This invention not only overcomes the inherent defects of existing RIP technology in the study of non-classical RNA binding proteins, but also achieves full-process optimization from high-specificity enrichment to high-quality sequencing sample preparation through the integrated innovation of methodology and reagent kit. It has outstanding advantages such as high specificity, high sensitivity, good reproducibility and simple operation, and has achieved significant technical progress and produced positive application effects. Attached Figure Description

[0015] Figure 1 For PrP C Agarose gel electrophoresis images of RNA enriched in Input group (1), RIP group (2), and IgG group (3) of C2C12 muscle cells. Gel concentration: 1%, voltage: 180V, electrophoresis time: 16min. M: marker.

[0016] Figure 2 For PrP C Agilent integrity assay results for RNA enriched in Input group (1), RIP group (2), and IgG group (3) of C2C12 muscle cells.

[0017] Figure 3 For PrP C RNA library diagram of RNA enriched in the Input and RIP groups in C2C12 muscle cells after library construction.

[0018] Figure 4 For PrP C RNA enriched by RIP assay in C2C12 muscle cells was sequenced by high-throughput miRNA-seq to identify PrP-related RNAs. C Volcano diagram of bound miRNAs (Control group on the left, WT PrP group on the right).

[0019] Figure 5 For PrP C Venn diagram of miRNAs specifically enriched by high-throughput sequencing miRNA-seq of RNA enriched by RIP experiment in C2C12 muscle cells (orange is the Control group, blue is the WT PrP group, and the overlapping part is the same miRNA enriched).

[0020] Figure 6 For PrP C Heatmap of miRNAs specifically enriched by high-throughput sequencing miRNA-seq of RNA enriched by RIP experiment in C2C12 muscle cells.

[0021] Figure 7 For PrP C Peak distribution of gene functional regions after high-throughput sequencing (miRNA-seq) of RNA enriched by RIP experiment in C2C12 muscle cells. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments, thereby making its advantages and various effects more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the invention.

[0023] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0024] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0025] The present application will now be described in detail with reference to specific embodiments and experimental data.

[0026] Example 1

[0027] This embodiment provides a case study for extracting RNA that specifically binds to prion proteins from muscle cells. The specific steps are as follows: (1) Prepare the relevant reagents.

[0028] RIP lysis buffer: Add Tris-HCl pH 7.4, NaCl, EDTA, NP-40, and glycerol to obtain the following components at various concentrations: 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol. Add RNase inhibitor, DTT, and protease inhibitor. After addition, the concentrations of the three are RNase inhibitor 400 U / ml, DTT 1 mM, and protease inhibitor 1×, respectively. Set aside for later use.

[0029] Wash buffer: Add Tris-HCl pH 7.4, NaCl, EDTA, NP-40, and glycerol to obtain the following components at various concentrations: 25 mM Tris-HCl pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol. Set aside. Take a portion of the Wash buffer and add RNase inhibitor, DTT, and protease inhibitor. After adding, the concentrations of the three are RNase inhibitor 400 U / ml, DTT 1 mM, and protease inhibitor 1×, respectively.

[0030] Proteinase K buffer was prepared in the following volume ratios: 10 μL 1 M Tris-HCl pH 7.4, 10 μL 5 M NaCl, 10 μL 0.5 M EDTA, 50 μL 10% SDS, 60 μL 20 mg / mL proteinase K solution, 10 μL 40 U / μL RNase inhibitor, 2 μL 0.5 M DTT, and 848 μL DEPC water. After preparation, the buffer was aliquoted into 150 μL portions and stored at -20 °C.

[0031] (2) RNA extraction steps: A. Muscle cell lysis: a. C2C12 muscle cells were cultured in 15 cm cell culture dishes and washed twice with pre-cooled PBS before the experiment;

[0032] b. Add DMEM to the cell culture dish;

[0033] c. Add 1% formaldehyde solution (prepared with PBS) (disperse the solution in the dish, then gently shake to mix), and let stand at room temperature for 10 minutes;

[0034] d. Add 1.25 M glycine solution (prepared with PBS) to the dish (disperse the solution in the dish and shake gently to mix), and let stand at room temperature for 5 min;

[0035] e. Wash twice with pre-cooled PBS;

[0036] f. Add pre-chilled PBS, scrape off cells on ice with a cell scraper, and aspirate into two EP tubes;

[0037] g. Centrifuge at 500 g for 5 min at 4 ℃, and discard the supernatant;

[0038] h. Add pre-cooled PBS, shake the wash dish (do not use a pipette to aspirate), and transfer it into the two EP tubes mentioned above;

[0039] i. Centrifuge at 4 ℃, 500 g for 5 min, and discard the supernatant;

[0040] j. Add 1 ml of RIP lysis buffer containing 400 U / ml RNase inhibitor, 1 mM DTT, and 1× protease inhibitor to each tube. Gently disperse the cells with a pipette and place on ice for lysis for 20 min (gently pipette once every 10 min).

[0041] k. Centrifuge the cell lysate at 12000 g for 10 min at 4 ℃;

[0042] 1. Take the supernatant and aspirate 100 μl from each tube into the same EP tube. That is, use 200 μl of cell lysis buffer as 10% Input. Add 1 ml of Trizol to the Input and store at -80 ℃ (as Input group).

[0043] B. Cell lysate-antibody-magnetic bead incubation: a. Take the supernatant, pipette 900 μl into two EP tubes, add 4 μl of antibody (8H4 1.8 mg / ml) to one tube as the RIP group (experimental group), and add 7.2 μl of mouse IgG (1 mg / ml) to the other tube as the IgG group (negative control group). Incubate in a rotary mixer at 4 ℃ for 5 h.

[0044] b. Take two EP tubes and add 75 μl of Protein A / G beads to each tube;

[0045] c. Wash twice with PBS, centrifuge at 1000 g for 5 min each time, and remove the supernatant;

[0046] d. Wash twice with Wash buffer, centrifuge at 1000 g for 5 min each time, and discard the supernatant; e. Wash once with Wash buffer containing 400 U / ml RNase inhibitor, 1 mM DTT, and 1× protease inhibitor, centrifuge at 1000 g for 5 min, and discard the supernatant;

[0047] f. Add the antibody-lysis buffer to the mixture and incubate in a rotary mixer at 4 °C for 3 h;

[0048] g. After incubation, centrifuge at 1000 g for 5 min at 4 ℃ and discard the supernatant;

[0049] h. Wash twice with Wash buffer containing 400 U / ml RNase inhibitor, 1 mM DTT, and 1× protease inhibitor. Centrifuge at 1000 g for 5 min each time, remove the supernatant, and obtain the magnetic bead incubators for the RIP group and IgG group, respectively.

[0050] C. Protein digestion: a. Remove the Input group EP tube containing Trizol from the -80 ℃ environment and thaw at room temperature. Add 150 μl of proteinase K buffer to each of the Input group EP tube and two EP tubes containing RIP group magnetic bead incubation and IgG group magnetic bead incubation, and incubate at 55 ℃ for 5 min.

[0051] b. After incubation, immediately place on ice to cool.

[0052] D. RNA extraction: a. Add 1 ml of Trizol to two EP tubes containing RIP group magnetic bead incubator and IgG group magnetic bead incubator, shake vigorously for 15 s, and let stand at room temperature for 10 min;

[0053] b. Add 200 μl of chloroform to every three EP tubes (RIP group, IgG group, Input group), shake vigorously for 15 s, and let stand at room temperature for 15 min;

[0054] c. Centrifuge all three EP tubes at 12000 g for 15 min at 4 ℃, and transfer the supernatant into three new EP tubes;

[0055] d. Add equal volumes of isopropanol to three new EP tubes, shake by hand for 15 s, then add 1 μl of Glycogen blue, let stand on ice for 10 min, and then place in a -80 ℃ freezer overnight to precipitate;

[0056] e. The following day, all samples were centrifuged at 16000 - 20000 g for 30 min at 4 ℃, and the supernatant was discarded;

[0057] f. Add 75% ethanol (prepared with DEPC water) to each tube and let stand on ice for 3 minutes;

[0058] g. All samples were centrifuged at 7500 g for 15 min at 4 ℃, and the supernatant was discarded;

[0059] h. All samples were centrifuged at 7500 g for 1 min at 4 ℃, and the residual alcohol on the tube wall was removed.

[0060] i. Lay flat on ice and let dry at room temperature for 10 minutes;

[0061] j. Add DEPC-treated water and RNase inhibitor to each tube. After measuring the concentration using a micro spectrophotometer, freeze the tubes in liquid nitrogen and promptly send them to a biotechnology company for high-throughput sequencing.

[0062] like Figure 1 The image shows agarose gel electrophoresis images of RNA extracted from the Input group, RIP group, and IgG group. It can be seen that the RNA extracted from the RIP group has very good integrity.

[0063] like Figure 2 The figure shows the Agilent integrity test results of RNA extracted from the Input group, RIP group, and IgG group. It can be seen that the RNA extracted from the RIP group has good integrity, high purity, and sufficient yield.

[0064] like Figure 3 As shown, the RNA libraries extracted from the Input group and the RIP group after library construction are RNA libraries (IgG was not constructed, and the Control group and WT PrP group are C2C12 cells treated by two different experimental methods, respectively). It can be seen that the RNA library data extracted from the RIP group after library construction is very ideal.

[0065] like Figure 4 As shown, this is a miRNA-seq dataset that identified PrP after high-throughput sequencing of RNA. C The volcano plot of the combined miRNAs shows that significant changes in miRNAs were extracted from both groups after significance analysis, indicating that it is feasible to use the RNA extracted by our RIP kit directly for high-throughput sequencing.

[0066] like Figure 5 As shown, the Venn diagram shows the specific enrichment of miRNAs after high-throughput sequencing of RNA using miRNA-seq. It can be seen that both groups were enriched with a large number of significantly changed miRNAs after significance analysis, which indicates that the RNA extracted by our RIP kit is feasible for high-throughput sequencing.

[0067] like Figure 6 The image shows a heatmap of miRNA-seq after high-throughput sequencing of RNA. It can be seen that miRNAs with high expression levels in the Input group and WT PrP group are not necessarily significantly enriched in the RIP. This indicates that the degree of miRNA enrichment is independent of its background expression level, thus demonstrating that PrP... C It can specifically enrich miRNAs during myoblast differentiation, which also reflects the specificity of RNA enrichment in our RIP kit.

[0068] like Figure 7The figure shows the peak distribution of gene functional regions after high-throughput sequencing of RNA using miRNA-seq. It can be seen that the RNA enriched by our RIP kit has good integrity and yield, and can peak in various gene functions after library construction.

[0069] Finally, it should be noted that the terms "comprising," "including," or any other variations are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0070] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. An RNA immunoprecipitation kit for specifically binding prion proteins in muscle cells, characterized in that, Includes RIP lysis buffer, Wash buffer, proteinase K buffer, and pre-use additives; The RIP lysis buffer comprises: 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol. The Wash buffer solution comprises: 25 mM Tris-HCl pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol; The proteinase K buffer comprises: 10 μL 1 M Tris-HCl pH 7.4, 10 μL 5 M NaCl, 10 μL 0.5 M EDTA, 50 μL 10% SDS, 60 μL 20 mg / mL proteinase K solution, 10 μL 40 U / μL RNase inhibitor, 2 μL 0.5 M DTT, and 848 μL DEPC water; The pre-use additives are added to RIP lysis buffer and Wash buffer before use. The pre-use additives include RNase inhibitors, DTT and protease inhibitors, and the concentrations of the three after addition are RNase inhibitor 400 U / ml, DTT 1 mM and protease inhibitor 1×, respectively.

2. The RNA immunoprecipitation kit as described in claim 1, characterized in that, The RNase inhibitor, DTT, and protease inhibitor are each packaged separately.

3. The RNA immunoprecipitation kit as described in claim 1, characterized in that, It also includes pre-packaged Protein A / G magnetic bead suspensions.

4. A method for RNA extraction using the RNA immunoprecipitation kit as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S10: Differentiation induction treatment: muscle cells were placed in a cell culture dish, DMEM culture medium was added, and then 1% formaldehyde solution prepared with PBS was added. After standing at room temperature for 10 min, 1.25 M glycine solution prepared with PBS was added dropwise and then stood at room temperature for 5 min. S20: Wash twice with pre-chilled PBS and then add pre-chilled PBS. Scrape cells with a cell scraper under ice and centrifuge to collect the cell pellet. Add RIP lysis buffer with pre-use additive to the cell pellet and lyse on ice for 20 minutes, gently pipetting every 10 minutes during the lysis. After lysis, centrifuge to collect the supernatant as the cell lysis buffer. S30: Take the cell lysate and place it in an EP tube, then add anti-PrP. C Monoclonal antibody 8H4 was incubated at 4°C for 5 hours by rotation. S40: Take a new EP tube, add Protein A / G magnetic beads to the EP tube, wash twice with PBS and discard the supernatant, then wash twice with Wash buffer and discard the supernatant, and finally wash once with Wash buffer containing the pre-use additive and discard the supernatant. S50: Add the antibody-lysis buffer mixture incubated in S30 to the treated Protein A / G magnetic beads in S40 and incubate at 4°C for 3 hours by rotation. After incubation, centrifuge and discard the supernatant, and wash the magnetic beads twice with Wash buffer containing pre-use additives, centrifuge and discard the supernatant. S60: Add proteinase K buffer to the magnetic beads and incubate at 55°C for 5 minutes to digest the protein. After incubation, immediately place on ice to cool. S70: Add Trizol to the digested sample, shake vigorously for 15 seconds, let stand at room temperature for 10 minutes, then add chloroform, shake vigorously again for 15 seconds, let stand at room temperature for 15 minutes; then centrifuge to collect the supernatant into a new EP tube, add an equal volume of isopropanol and shake for 15 seconds, then add 1 μL of Glycogen blue, let stand on ice for 10 minutes, then incubate overnight at -80℃ to precipitate RNA, centrifuge the next day and discard the supernatant; S80: Wash the RNA precipitate twice with 75% ethanol under ice bath; after air drying at room temperature for 10 minutes, dissolve the RNA in DEPC water, add RNase inhibitor, determine the concentration, and then freeze in liquid nitrogen for later use.

5. The RNA extraction method as described in claim 4, characterized in that, In step S10, the muscle cells are washed twice with pre-cooled PBS before being placed in the cell culture dish.

Images