CN111458518B - Development and application of simple RNA binding protein immunoprecipitation kit - Google Patents
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Abstract
The invention relates to the field of molecular biology, in particular to a kit for detecting RNA binding protein immunoprecipitation and a method for detecting RNA binding protein immunoprecipitation by using the same. The kit comprises TBST buffer solution, RIPA lysate, protein G/A magnetic beads and RNase inhibitor. The TBST buffer comprises: 20mM Tris-HCl, 500mM NaCl, 0.05% Tween 20 (pH 7.5); the RIPA lysate comprises: 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.1% SDS; the Protein G/A magnetic beads are Protein G/A agarose beads. The method for detecting the RNA binding protein by using the kit has the advantages of simple operation, low cost and high efficiency, is suitable for large-scale popularization and use, and can save a great amount of time and expense for scientific researchers.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a kit for detecting RNA binding protein immunoprecipitation and a method for detecting RNA binding protein immunoprecipitation by using the same.
Background
RNA binding proteins (RNA binding proteins, RBPs), a class of proteins that interact directly with RNA molecules, RBPs are key components in metabolism that can be involved in various layers of RNA post-transcriptional modification, such as splicing of RNA exons, polyadenylation, transport from the nucleoplasm to subcellular, intracellular localization, translation, degradation, and degeneration. Intracellular RNAs are not independent but exist in the form of more functional ribonucleoprotein complexes (RNPs). RNP is a functional unit of RBP formed by binding to a coding or non-coding protein. RBP can bind RNA stably both in time and space.
The regulation of gene expression plays an important role in the growth, differentiation, stress response and other aspects of cells in organisms. However, in addition to the regulation of transcription by transcription factors, post-transcriptional regulation of cells plays an important role in physiological processes, including the specific and rapid regulation of mRNA transcription rate by RNA-binding proteins. RNA co-immunoprecipitation (RNA immunoprecipitating, RIP) is a technique for studying the relationship between RNA transcripts specifically binding to RNA binding proteins in vivo by the method of co-immunoprecipitation, based on antigen-antibody reactions.
RIP technology is the technology for researching the combination condition of RNA and protein in cells, is a powerful tool for knowing the dynamic process of a post-transcriptional regulatory network, and can help to find the regulatory targets of non-coding RNA. RIP technology uses antibodies to target proteins to precipitate corresponding RNA-protein complexes, which are then separated and purified to analyze the RNA bound to the complexes. RIP can be seen as a similar application of the commonly used chromatin immunoprecipitation ChIP technique, but since the study object is an RNA-protein complex rather than a DNA-protein complex, the optimal conditions for RIP experiments are not much the same as ChIP experiments (e.g., complex does not need to be immobilized, reagents and antibodies in the RIP reaction system cannot contain rnase at all, antibodies need to be validated by RIP experiments, etc.). The downstream binding microarray technology of the RIP technology is called RIP-Chip, helping to understand the RNA changes at the overall level of cancer and other diseases with higher throughput.
95% of the human genome does not encode genes, but rather produces large amounts of non-coding RNA, with genes that actually encode proteins representing only about 2% of the total human genome. These non-coding RNAs play important regulatory roles in various stages of growth and development of life, are closely related to various lesions such as AIDS, leukemia, diabetes, deformity and the like, and are involved in stem cell and epigenetic regulation. While RNA-protein complexes drive posttranscriptional regulation of gene expression in almost all cellular processes, including splicing (splicing), nuclear export (nuclear export), mRNA stability, and protein translation processes, knowledge of gene regulation is therefore dependent on determining changes in RNA binding during these processes. Thus, RNA research is also being appreciated by more and more scientists, and has become a hot field in life science research, while RIP technology is becoming a routine method in RNA research, helping to understand the increasingly focused post-transcriptional regulatory networks. At present, the RIP experiment is mostly operated by adopting a kit method, but the experiment cost is very high (about 8000 yuan of the kit for detecting 12 samples at home and abroad) and the economic burden of researchers is greatly increased. Therefore, development of a method for rapidly and accurately detecting RNA binding protein has been a new topic to be solved.
Disclosure of Invention
Aiming at the problems, the invention provides a kit for detecting RNA binding protein immunoprecipitation by combining a molecular biology experimental technology according to an immunological principle, and a method for detecting RNA binding protein immunoprecipitation by using the kit. The experimental cost is greatly reduced, and the kit has the advantages of good detection effect on various genes and various cells compared with the existing similar kit.
In order to achieve the above object, the present invention adopts the following technical scheme.
A kit for detecting immunoprecipitation of an RNA binding Protein, the kit comprising TBST buffer, RIPA lysate, protein G/a beads, RNase inhibitor.
Further, the TBST buffer comprises: 20mM Tris-HCl, 500mM NaCl, 0.05% Tween 20 (pH 7.5).
Further, the RIPA lysate comprises 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.1% SDS.
Further, the Protein G/A magnetic beads are Protein G/A agarose beads.
A method for immunoprecipitation detection of an RNA binding protein using a kit for detecting an RNA binding protein, specifically comprising the following steps.
Rna binding protein immunopurification process.
1) The cells in the flask were washed twice with cold PBS.
2) 1500rpm, centrifugation for 5min, removal of supernatant and collection of cells into EP tubes.
3) The RIPA lysate was added at 300ul, the cells resuspended, and after homogenization, the cells were blown up overnight at 4 ℃.
4) The next day, centrifugation was carried out at 12000rpm for 20 minutes at 4℃and the supernatant was transferred to new EP tubes, each of which was filled with 150ul of cell lysate.
5) lgG and 4ug of the target antibody were added to the cell lysates, respectively, and incubated at 4℃for 4 hours.
6) Protein G/A agarose beads were added in 20ul, respectively, and the mixture was shaken at room temperature for 4 hours.
7) 10000rpm,4 ℃ for 2min, and discarding the supernatant.
8) TBST liquid was repeatedly purged 2 times, spun at 10000rpm at 4℃for 1min, and the supernatant was discarded.
Rna purification process.
1) To the above precipitate, 1ml of Trizol was added, and 200ul of chloroform (Trizol: chloroform = 5: 1) Shaking vigorously for 15 seconds, and standing at room temperature for 5 minutes after the solution is fully emulsified.
2) Centrifugation at 12,000rpm for 20 min at 4℃and transfer of the supernatant to another new EP tube.
3) Adding equal volume of isopropanol into the supernatant, reversing the EP tube upside down, fully and uniformly mixing, and standing for 10 minutes at room temperature; the supernatant was discarded by centrifugation at 12,000rpm for 20 minutes at 4 ℃.
4) Adding lml of 75% ethanol along the tube wall, washing the EP tube wall upside down, centrifuging at 10000rpm at 4deg.C for 10 min, discarding ethanol, drying at room temperature for 5-10 min, and adding appropriate amount of 10ul RNase-free water.
3. cDNA was synthesized by reverse transcription.
4.Real-time PCR。
Compared with the prior art, the invention has the following beneficial effects.
The method for detecting the RNA binding protein by using the kit has the advantages of simple operation, low cost and high efficiency, is suitable for large-scale popularization and use, and can save a great amount of time and expense for scientific researchers.
Drawings
FIG. 1 is an amplification curve after amplification using ABI 7500.
FIG. 2 is a melting curve after amplification using ABI 7500.
Fig. 3 shows the results of the target protein group CT value-IgG group CT value= Δ CT.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples illustrate the invention in detail, but are not intended to limit the scope of the invention. This example uses routine experimentation, which is familiar to those skilled in the art, and can be performed according to this example using instructions provided by the materials manufacturer.
Examples the kit of the invention is used for the immunoprecipitation detection of RNA binding proteins.
Rna binding protein immunopurification process.
1) The cells in the flask were washed twice with cold PBS.
2) 1500rpm, centrifugation for 5min, removal of supernatant and collection of cells into EP tubes.
3) The RIPA lysate was added at 300ul, the cells resuspended, and after homogenization, the cells were blown up overnight at 4 ℃.
4) The next day, centrifugation was carried out at 12000rpm for 20 minutes at 4℃and the supernatant was transferred to new EP tubes, each of which was filled with 150ul of cell lysate.
5) lgG and 4ug of the target antibody were added to the cell lysates, respectively, and incubated at 4℃for 4 hours.
6) Protein G/A agarose beads were added in 20ul, respectively, and the mixture was shaken at room temperature for 4 hours.
7) 10000rpm,4 ℃ for 2min, and discarding the supernatant.
8) TBST liquid was repeatedly purged 2 times, 10,000rpm, centrifuged at 4℃for 1min, and the supernatant was discarded.
Rna purification process.
1) To the above precipitate, 1ml of Trizol was added, and 200ul of chloroform (Trizol: chloroform = 5: 1) Shaking vigorously for 15 seconds, and standing at room temperature for 5 minutes after the solution is fully emulsified.
2) Centrifugation at 12000rpm for 20 min at 4℃and transfer of the supernatant to another new EP tube.
3) Adding equal volume of isopropanol into the supernatant, reversing the EP tube upside down, fully and uniformly mixing, and standing for 10 minutes at room temperature; the supernatant was discarded by centrifugation at 12000rpm for 20 minutes at 4 ℃.
4) Adding lml of 75% ethanol along the tube wall, washing the EP tube wall upside down, centrifuging at 10,000rpm at 4deg.C for 10 min, discarding ethanol, drying at room temperature for 5-10 min, and adding appropriate amount of 10ul RNase-free water.
3. cDNA was synthesized by reverse transcription.
The cDNA obtained was reverse transcribed using the GoScript reverse transcription system (A5000, A5001) according to the following procedure.
1) A certain amount of template RNA is taken and added into a primer.
2) The mixture of template RNA and primer was subjected to pre-denaturation at 70℃for 5min, and after completion, the mixture was taken out and placed on ice.
3) RT-Mix was formulated and added to each sample tube.
4) Reverse transcription procedure was set up, including three steps of annealing, extension, and reverse transcriptase inactivation (annealing at 25℃for 5min, extension at 42℃for 60min, inactivation at 70℃for 15min, and 4℃++ infinity). After the completion of the procedure, cDNA was obtained.
4.Real-time PCR。
1) The PCR reaction mixture was prepared as follows (the preparation of the reaction mixture may be carried out at room temperature).
2)ABI7500Real-Time PCR System the following settings were used.
The PCR standard amplification procedure performed in the two-step method is shown in Table 1.
Table 1 two-step method PCR standard amplification procedure was performed.
3) Export data, real time PCR results with 2 -△△CT The method was used for analysis.
4) The result was calculated as target proteome CT-IgG proteome ct=.DELTA.DELTA.CT. The results are shown in FIG. 3.
Claims (1)
1. A kit for detecting immunoprecipitation of an RNA binding Protein, comprising a TBST buffer, RIPA lysate, protein G/a sepharose beads, an RNase inhibitor; the method for detecting the RNA binding protein immunoprecipitation by using the kit specifically comprises the following steps:
step 1, RNA binding protein immune purification process:
1) Washing the cells in the flask twice with cold PBS;
2) Centrifugation at 1500rpm for 5min, discarding supernatant and collecting cells into EP tube;
3) Adding 300uL of RIPA lysate, re-suspending cells, and blowing uniformly, and then standing at 4 ℃ overnight; the RIPA lysate comprises: 50mM Tris-HCl, 150mM NaCl, 1% NP-40, 0.1% SDS at pH 7.4;
4) The next day, centrifugation at 12,000rpm at 4℃for 20 min, taking supernatant, transferring to new EP tubes, and sub-packaging 150uL of cell lysate per tube;
5) lgG and 4ug of target antibody are added into the cell lysate respectively, and the cells are incubated for 4 hours at 4 ℃;
6) Protein G/A agarose beads 20uL are added respectively, and the mixture is shaken for 4 hours at room temperature;
7) Centrifuging at 10000rpm at 4deg.C for 2min, and discarding supernatant;
8) Repeatedly purging with TBST buffer solution for 2 times, centrifuging at 10,000rpm and 4deg.C for 1min, and discarding supernatant to obtain precipitate; the TBST buffer comprises: 20mM Tris-HCl, 500mM NaCl, 0.05% Tween 20 at pH 7.5;
step 2, RNA purification process:
1) To the above precipitate was added 1mL of Trizol, and 200uL of chloroform, trizol: chloroform = 5:1, shaking vigorously for 15 seconds,
standing for 5 minutes at room temperature after the solution is fully emulsified;
2) Centrifuging at 12000rpm for 20 min at 4deg.C, and transferring the supernatant to another new EP tube;
3) Adding equal volume of isopropanol into the supernatant, reversing the EP tube upside down, fully and uniformly mixing, and standing for 10 minutes at room temperature; centrifuging at 12000rpm for 20 min at 4deg.C, and discarding supernatant;
4) Adding lmL ethanol with concentration of 75% along the tube wall, washing the EP tube wall upside down, centrifuging at 10,000rpm at 4deg.C for 10 min, discarding ethanol, drying at room temperature for 5-10 min, and adding appropriate amount of 10uL RNase-free water;
step 3, synthesizing cDNA by reverse transcription;
and 4, real-time PCR.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003099957A1 (en) * | 2002-05-22 | 2003-12-04 | Duke University | Rnp immunoprecipitation assay |
| CN103232998A (en) * | 2013-04-22 | 2013-08-07 | 中国药科大学 | Kit for separating RNA (ribonucleic acid) bound in RNA binding protein |
| CN107674870A (en) * | 2016-08-01 | 2018-02-09 | 武汉生命之美科技有限公司 | A kind of method of the target RNA sequence of rna binding protein in improved identification of cell sample |
| CN109337901A (en) * | 2018-10-31 | 2019-02-15 | 上海微斯生物科技有限公司 | The chromosome co-immunoprecipitation method and kit of DNA binding protein in eukaryocyte |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003099957A1 (en) * | 2002-05-22 | 2003-12-04 | Duke University | Rnp immunoprecipitation assay |
| CN103232998A (en) * | 2013-04-22 | 2013-08-07 | 中国药科大学 | Kit for separating RNA (ribonucleic acid) bound in RNA binding protein |
| CN107674870A (en) * | 2016-08-01 | 2018-02-09 | 武汉生命之美科技有限公司 | A kind of method of the target RNA sequence of rna binding protein in improved identification of cell sample |
| CN109337901A (en) * | 2018-10-31 | 2019-02-15 | 上海微斯生物科技有限公司 | The chromosome co-immunoprecipitation method and kit of DNA binding protein in eukaryocyte |
Non-Patent Citations (1)
| Title |
|---|
| 田素雯 等.运用紫外交联免疫沉淀技术构建RNA 结合蛋白的靶基因文库.基因组学与应用生物学.2017,第36卷(第6期),第2152-2156 页. * |
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