Experimental device for screening RNA (Ribonucleic Acid) through RNA binding protein immunoprecipitation

Abstract

The invention discloses an experimental device for screening RNA through RNA binding protein immunoprecipitation, and particularly relates to the field of biology, the experimental device comprises a sample processing unit, an immunoprecipitation unit, an RNA extraction unit and a detection analysis unit, the sample processing unit is connected with the immunoprecipitation unit, the immunoprecipitation unit is connected with the RNA extraction unit, and the detection analysis unit is connected with the RNA extraction unit. The RNA extraction unit is connected with the detection and analysis unit; the sample treatment unit is used for treating the liver cancer cell sample; the immunoprecipitation unit is used for capturing RNA binding protein; the RNA extraction unit is used for releasing the combined RNA and extracting the RNA; the detection and analysis unit is used for analyzing RNA, and the operation of the sample treatment unit specifically comprises the following steps: step a1, taking primary hepatoma carcinoma cells separated and cultured from tumor tissues of a hepatoma carcinoma patient as hepatoma carcinoma cell samples; a2, adding the liver cancer cell sample into a stirrer for stirring; the RNA can be quickly screened, the operation is relatively convenient, and the accuracy is high.

Description

Technical Field

[0001] The present invention relates to the biological field, and more specifically to an experimental device for RNA binding protein immunoprecipitation and RNA screening. Background Art

[0002] Liver cancer is a serious malignant tumor, and its occurrence and development involve abnormal regulation of multiple genes and signaling pathways. By screening RNA-binding proteins in liver cancer cells, we can gain a deeper understanding of the mechanism of action of RNA-binding proteins in the occurrence, development, and metastasis of liver cancer, and provide new targets for the diagnosis and treatment of liver cancer. Certain RNA-binding proteins may be specifically expressed in liver cancer cells or closely related to the progression of liver cancer, and are expected to become biomarkers for liver cancer. For example, detecting the expression level of specific RNA-binding proteins in blood or tissues can be used for early diagnosis, prognosis assessment, and treatment monitoring of liver cancer.

[0003] RNA-binding protein immunoprecipitation screening RNA is a powerful technology for studying the interaction between RNA and protein. RNA-binding proteins play an important role in the regulation of gene expression. Studying the interaction between RNA-binding proteins and RNA is crucial to understanding the regulatory mechanism of gene expression. At present, the commonly used RNA-binding protein immunoprecipitation (RIP) technology can specifically enrich RNA bound to specific RNA-binding proteins. However, the existing experimental methods and devices have some shortcomings in the operation process, such as complex operation, low efficiency, and low accuracy.

[0004] In order to solve the above problems, a technical solution is now provided. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, an embodiment of the present invention provides an experimental device for RNA-binding protein immunoprecipitation screening RNA to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] An experimental device for RNA binding protein immunoprecipitation screening RNA, comprising a sample processing unit, an immunoprecipitation unit, an RNA extraction unit and a detection and analysis unit, wherein the sample processing unit is connected to the immunoprecipitation unit, the immunoprecipitation unit is connected to the RNA extraction unit, and the RNA extraction unit is connected to the detection and analysis unit;

[0008] The sample processing unit is used for processing liver cancer cell samples;

[0009] The immunoprecipitation unit is used to capture RNA-binding proteins;

[0010] The RNA extraction unit is used to release the bound RNA and extract the RNA;

[0011] The detection and analysis unit is used to analyze RNA.

[0012] In a preferred embodiment, the sample processing unit operation specifically includes the following:

[0013] Step a1, using primary liver cancer cells isolated and cultured from tumor tissues of liver cancer patients as liver cancer cell samples;

[0014] Step a2, adding the liver cancer cell sample into a blender for blending;

[0015] Step a3, controlling the temperature of the liver cancer cell sample during stirring, and setting the temperature to 36-38°C.

[0016] It should be added that using liver cancer cells as biological samples for RNA binding protein screening is of great significance for in-depth research on the pathogenesis of liver cancer, discovery of new biomarkers and development of therapeutic drugs. However, there are also some challenges in the experimental process, which requires the comprehensive use of multiple technical means and analytical methods to obtain accurate and reliable results.

[0017] In a preferred embodiment, the immunoprecipitation unit operation specifically includes the following:

[0018] Step b1, lysing the liver cancer cell sample to release RNA binding protein;

[0019] Step b2, add magnetic beads, set the temperature to 3-5°C, and set the time to 60-70min;

[0020] Step b3, separating and washing the magnetic beads to capture RNA binding proteins.

[0021] In a preferred embodiment, the RNA extraction unit specifically includes the following contents:

[0022] Step c1, cleaving the RNA binding protein;

[0023] Step c2, transferring the cleaved RNA binding protein to an adsorption column;

[0024] Step c3, adding washing liquid to the adsorption column and centrifuging to remove residual impurities;

[0025] Step c4, perform elution treatment, collect and extract RNA after centrifugation.

[0026] It should be added that magnetic beads can specifically capture RNA-binding proteins, thereby achieving the separation and enrichment of target proteins and their bound RNA, laying the foundation for subsequent RNA extraction and analysis.

[0027] In a preferred embodiment, the detection and analysis unit operation includes the following specific contents:

[0028] Step d1, testing the concentration, purity, integrity and specific RNA of the extracted RNA;

[0029] Step d2, respectively obtaining concentration parameters, purity parameters, integrity parameters and specific RNA parameters;

[0030] Step d3, marking the concentration parameter as ND, the purity parameter as CD, the integrity parameter as WZX, and the specific RNA parameter as TD;

[0031] In step d4, concentration parameters, purity parameters, integrity parameters and specific RNA parameters are normalized.

[0032] It should be added that the concentration, purity, integrity and detection of specific RNA should be considered comprehensively to determine whether the extracted RNA is suitable for subsequent experimental needs, and appropriate measures should be taken according to the results for optimization or re-experimentation.

[0033] In a preferred embodiment, the normalization formula is:

[0034]

[0035] Among them, PG is the evaluation parameter;

[0036] a1, a2, a3, and a4 are the proportional coefficients of concentration parameter, purity parameter, integrity parameter, and specific RNA parameter, respectively.

[0037] In a preferred embodiment, an evaluation parameter signal threshold is set, the evaluation parameter signal threshold is marked as PG1, and the evaluation parameter PG is compared with the evaluation parameter signal threshold PG1;

[0038] If PG<PG1, it means normal;

[0039] If PG≥PG1, it indicates an exception and generates abnormal data.

[0040] In a preferred embodiment, statistics are collected on abnormal data to generate a pass rate.

[0041] The technical effects and advantages of the experimental device for RNA binding protein immunoprecipitation and RNA screening of the present invention are as follows:

[0042] 1. By processing liver cancer cell samples, capturing RNA binding proteins after processing, then releasing the bound RNA and extracting the RNA, and finally analyzing the RNA, the RNA can be screened quickly, which is convenient to operate and has high accuracy;

[0043] 2. By normalizing the concentration parameters, purity parameters, integrity parameters and specific RNA parameters to obtain evaluation parameters, and then analyzing the evaluation parameters, the analysis efficiency can be improved and RNA can be screened quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The present invention is a schematic diagram of the structure of an experimental device for RNA-binding protein immunoprecipitation and RNA screening. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] Figure 1 Provided is an experimental device for RNA binding protein immunoprecipitation screening RNA, comprising a sample processing unit, an immunoprecipitation unit, an RNA extraction unit and a detection and analysis unit, wherein the sample processing unit is connected to the immunoprecipitation unit, the immunoprecipitation unit is connected to the RNA extraction unit, and the RNA extraction unit is connected to the detection and analysis unit;

[0048] The sample processing unit is used for processing liver cancer cell samples;

[0049] The immunoprecipitation unit is used to capture RNA-binding proteins;

[0050] The RNA extraction unit is used to release the bound RNA and extract the RNA;

[0051] The detection and analysis unit is used to analyze RNA.

[0052] Specifically, the sample processing unit operation includes the following:

[0053] Step a1, using primary liver cancer cells isolated and cultured from tumor tissues of liver cancer patients as liver cancer cell samples;

[0054] Step a2, adding the liver cancer cell sample into a blender for blending;

[0055] Step a3, controlling the temperature of the liver cancer cell sample during stirring, and setting the temperature to 36°C.

[0056] Specifically, the immunoprecipitation unit operation includes the following:

[0057] Step b1, lysing the liver cancer cell sample to release RNA binding protein;

[0058] Step b2, add magnetic beads, set the temperature to 3°C, and set the time to 60 min;

[0059] Step b3, separating and washing the magnetic beads to capture RNA binding proteins.

[0060] Specifically, the RNA extraction unit includes the following contents:

[0061] Step c1, cleaving the RNA binding protein;

[0062] Step c2, transferring the cleaved RNA binding protein to an adsorption column;

[0063] Step c3, adding washing liquid to the adsorption column and centrifuging to remove residual impurities;

[0064] Step c4, perform elution treatment, collect and extract RNA after centrifugation.

[0065] Specifically, the operation of the detection and analysis unit includes the following specific contents:

[0066] Step d1, testing the concentration, purity, integrity and specific RNA of the extracted RNA;

[0067] Step d2, respectively obtaining concentration parameters, purity parameters, integrity parameters and specific RNA parameters;

[0068] Step d3, marking the concentration parameter as ND, the purity parameter as CD, the integrity parameter as WZX, and the specific RNA parameter as TD;

[0069] In step d4, concentration parameters, purity parameters, integrity parameters and specific RNA parameters are normalized.

[0070] Specifically, the normalization formula is:

[0071] Among them, PG is the evaluation parameter;

[0072] a1, a2, a3, and a4 are the proportional coefficients of concentration parameter, purity parameter, integrity parameter, and specific RNA parameter, respectively.

[0073] Setting an evaluation parameter signal threshold, marking the evaluation parameter signal threshold as PG1, and comparing the evaluation parameter PG with the evaluation parameter signal threshold PG1;

[0074] If PG<PG1, it means normal;

[0075] If PG≥PG1, it indicates an exception and generates abnormal data.

[0076] Statistics are collected on abnormal data to generate the pass rate.

[0077] Example 2

[0078] Figure 1 Provided is an experimental device for RNA binding protein immunoprecipitation screening RNA, comprising a sample processing unit, an immunoprecipitation unit, an RNA extraction unit and a detection and analysis unit, wherein the sample processing unit is connected to the immunoprecipitation unit, the immunoprecipitation unit is connected to the RNA extraction unit, and the RNA extraction unit is connected to the detection and analysis unit;

[0079] The sample processing unit is used for processing liver cancer cell samples;

[0080] The immunoprecipitation unit is used to capture RNA-binding proteins;

[0081] The RNA extraction unit is used to release the bound RNA and extract the RNA;

[0082] The detection and analysis unit is used to analyze RNA.

[0083] Specifically, the sample processing unit operation includes the following:

[0084] Step a1, using primary liver cancer cells isolated and cultured from tumor tissues of liver cancer patients as liver cancer cell samples;

[0085] Step a2, adding the liver cancer cell sample into a blender for blending;

[0086] Step a3, controlling the temperature of the liver cancer cell sample during stirring, and setting the temperature to 37°C.

[0087] Specifically, the immunoprecipitation unit operation includes the following:

[0088] Step b1, lysing the liver cancer cell sample to release RNA binding protein;

[0089] Step b2, add magnetic beads, set the temperature to 4°C, and set the time to 65 min;

[0090] Step b3, separating and washing the magnetic beads to capture RNA binding proteins.

[0091] Specifically, the RNA extraction unit includes the following contents:

[0092] Step c1, cleaving the RNA binding protein;

[0093] Step c2, transferring the cleaved RNA binding protein to an adsorption column;

[0094] Step c3, adding washing liquid to the adsorption column and centrifuging to remove residual impurities;

[0095] Step c4, perform elution treatment, collect and extract RNA after centrifugation.

[0096] Specifically, the operation of the detection and analysis unit includes the following specific contents:

[0097] Step d1, testing the concentration, purity, integrity and specific RNA of the extracted RNA;

[0098] Step d2, respectively obtaining concentration parameters, purity parameters, integrity parameters and specific RNA parameters;

[0099] Step d3, marking the concentration parameter as ND, the purity parameter as CD, the integrity parameter as WZX, and the specific RNA parameter as TD;

[0100] In step d4, concentration parameters, purity parameters, integrity parameters and specific RNA parameters are normalized.

[0101] Specifically, the normalization formula is:

[0102] Among them, PG is the evaluation parameter;

[0103] a1, a2, a3, and a4 are the proportional coefficients of concentration parameter, purity parameter, integrity parameter, and specific RNA parameter, respectively.

[0104] Setting an evaluation parameter signal threshold, marking the evaluation parameter signal threshold as PG1, and comparing the evaluation parameter PG with the evaluation parameter signal threshold PG1;

[0105] If PG<PG1, it means normal;

[0106] If PG≥PG1, it indicates an exception and generates abnormal data.

[0107] Statistics are collected on abnormal data to generate the pass rate.

[0108] Example 3

[0109] Figure 1Provided is an experimental device for RNA binding protein immunoprecipitation screening RNA, comprising a sample processing unit, an immunoprecipitation unit, an RNA extraction unit and a detection and analysis unit, wherein the sample processing unit is connected to the immunoprecipitation unit, the immunoprecipitation unit is connected to the RNA extraction unit, and the RNA extraction unit is connected to the detection and analysis unit;

[0110] The sample processing unit is used for processing liver cancer cell samples;

[0111] The immunoprecipitation unit is used to capture RNA-binding proteins;

[0112] The RNA extraction unit is used to release the bound RNA and extract the RNA;

[0113] The detection and analysis unit is used to analyze RNA.

[0114] Specifically, the sample processing unit operation includes the following:

[0115] Step a1, using primary liver cancer cells isolated and cultured from tumor tissues of liver cancer patients as liver cancer cell samples;

[0116] Step a2, adding the liver cancer cell sample into a blender for blending;

[0117] Step a3, controlling the temperature of the liver cancer cell sample during stirring, and setting the temperature to 38°C.

[0118] Specifically, the immunoprecipitation unit operation includes the following:

[0119] Step b1, lysing the liver cancer cell sample to release RNA binding protein;

[0120] Step b2, adding magnetic beads, setting the temperature to 5°C, and the time to 70 min;

[0121] Step b3, separating and washing the magnetic beads to capture RNA binding proteins.

[0122] Specifically, the RNA extraction unit includes the following contents:

[0123] Step c1, cleaving the RNA binding protein;

[0124] Step c2, transferring the cleaved RNA binding protein to an adsorption column;

[0125] Step c3, adding washing liquid to the adsorption column and centrifuging to remove residual impurities;

[0126] Step c4, perform elution treatment, collect and extract RNA after centrifugation.

[0127] Specifically, the operation of the detection and analysis unit includes the following specific contents:

[0128] Step d1, testing the concentration, purity, integrity and specific RNA of the extracted RNA;

[0129] Step d2, respectively obtaining concentration parameters, purity parameters, integrity parameters and specific RNA parameters;

[0130] Step d3, marking the concentration parameter as ND, the purity parameter as CD, the integrity parameter as WZX, and the specific RNA parameter as TD;

[0131] In step d4, concentration parameters, purity parameters, integrity parameters and specific RNA parameters are normalized.

[0132] Specifically, the normalization formula is:

[0133] Among them, PG is the evaluation parameter;

[0134] a1, a2, a3, and a4 are the proportional coefficients of concentration parameter, purity parameter, integrity parameter, and specific RNA parameter, respectively.

[0135] Setting an evaluation parameter signal threshold, marking the evaluation parameter signal threshold as PG1, and comparing the evaluation parameter PG with the evaluation parameter signal threshold PG1;

[0136] If PG<PG1, it means normal;

[0137] If PG≥PG1, it indicates an exception and generates abnormal data.

[0138] Statistics are collected on abnormal data to generate the pass rate.

[0139] In Examples 1 to 3, each example processes 20 groups of liver cancer cell samples, and the number of abnormal data and the qualified rate are counted. The results are shown in the following table:

[0140] Example Number of abnormal data Pass rate Example 1 2 90％ Example 2 3 85％ Example 3 1 95％

[0141] It can be seen that the experimental device of the present invention can be used to screen RNA in liver cancer cell samples, with a high pass rate and accuracy after screening, and Example 3 is the best example.

[0142] The above formulas are all dimensionless and numerical calculations. The formula is a formula for the most recent real situation obtained by collecting a large amount of data and performing software simulation. The preset parameters and thresholds in the formula are set by technicians in this field according to actual conditions.

[0143] The above embodiments can be implemented in whole or in part by software, hardware, firmware or any other combination. When implemented using software, the above embodiments can be implemented in whole or in part in the form of a computer program product. The computer program product includes one or more computer instructions or computer programs. When the computer instructions or computer programs are loaded or executed on a computer, the process or function described in the embodiment of the present application is generated in whole or in part. The computer can be a general-purpose computer, a special-purpose computer, a computer network, or other programmable devices. The computer instructions can be stored in a computer-readable storage medium, or transmitted from one computer-readable storage medium to another computer-readable storage medium. For example, the computer instructions can be transmitted from one website site, computer, server or data center to another website site, computer, server or data center by wired (e.g., infrared, wireless, microwave, etc.). The computer-readable storage medium can be any available medium that a computer can access or a data storage device such as a server or data center that contains one or more available media sets. The available medium can be a magnetic medium (e.g., a floppy disk, a hard disk, a tape), an optical medium (e.g., a DVD), or a semiconductor medium. The semiconductor medium can be a solid-state hard disk.

[0144] Those of ordinary skill in the art will appreciate that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0145] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned embodiments and will not be repeated here.

[0146] In the several embodiments provided in the present application, it should be understood that the disclosed systems and devices can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0147] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, and may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0148] In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit.

[0149] If the functions are implemented in the form of software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present application can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product is stored in a storage medium and includes several instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps described in each embodiment of the present application. The aforementioned storage medium includes: various media that can store program codes, such as a USB flash drive, a mobile hard disk, a read-only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk.

[0150] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

[0151] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An experimental device for RNA binding protein immunoprecipitation screening RNA, characterized in that: It includes a sample processing unit, an immunoprecipitation unit, an RNA extraction unit and a detection and analysis unit, wherein the sample processing unit is connected to the immunoprecipitation unit, the immunoprecipitation unit is connected to the RNA extraction unit, and the RNA extraction unit is connected to the detection and analysis unit; The sample processing unit is used for processing liver cancer cell samples; The immunoprecipitation unit is used to capture RNA-binding proteins; The RNA extraction unit is used to release the bound RNA and extract the RNA; The detection and analysis unit is used to analyze RNA.

2. The experimental device for RNA-binding protein immunoprecipitation screening RNA according to claim 1, characterized in that: The sample processing unit operation specifically includes the following: Step a1, using primary liver cancer cells isolated and cultured from tumor tissues of liver cancer patients as liver cancer cell samples; Step a2, adding the liver cancer cell sample into a blender for blending; Step a3, controlling the temperature of the liver cancer cell sample during stirring, and setting the temperature to 36-38°C.

3. The experimental device for RNA-binding protein immunoprecipitation screening RNA according to claim 1, characterized in that: The immunoprecipitation unit run specifically includes the following: Step b1, lysing the liver cancer cell sample to release RNA binding protein; Step b2, add magnetic beads, set the temperature to 3-5°C, and set the time to 60-70min; Step b3, separating and washing the magnetic beads to capture RNA binding proteins.

4. The experimental device for RNA binding protein immunoprecipitation screening RNA according to claim 1, characterized in that: The RNA extraction unit specifically includes the following: Step c1, cleaving the RNA binding protein; Step c2, transferring the cleaved RNA binding protein to an adsorption column; Step c3, adding washing liquid to the adsorption column and centrifuging to remove residual impurities; Step c4, perform elution treatment, collect and extract RNA after centrifugation.

5. The experimental device for RNA-binding protein immunoprecipitation screening RNA according to claim 1, characterized in that: The operation of the detection and analysis unit includes the following specific contents: Step d1, testing the concentration, purity, integrity and specific RNA of the extracted RNA; Step d2, respectively obtaining concentration parameters, purity parameters, integrity parameters and specific RNA parameters; Step d3, mark the concentration parameter as ND, the purity parameter as CD, the integrity parameter as WZX, and the specific RNA parameter as TD; In step d4, concentration parameters, purity parameters, integrity parameters and specific RNA parameters are normalized.

6. The experimental device for RNA-binding protein immunoprecipitation screening of RNA according to claim 5, characterized in that: The formula for normalization is: Among them, PG is the evaluation parameter; a1, a2, a3, and a4 are the proportional coefficients of concentration parameter, purity parameter, integrity parameter, and specific RNA parameter, respectively.

7. The experimental device for RNA-binding protein immunoprecipitation screening of RNA according to claim 6, characterized in that: Setting an evaluation parameter signal threshold, marking the evaluation parameter signal threshold as PG1, and comparing the evaluation parameter PG with the evaluation parameter signal threshold PG1; If PG<PG1, it means normal; If PG≥PG1, it indicates an exception and generates abnormal data.

8. The experimental device for RNA-binding protein immunoprecipitation screening of RNA according to claim 7, characterized in that: Statistics are collected on abnormal data to generate the pass rate.

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