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LncRNA data deep mining tool
August 15, 2025
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Experimental techniques such as RIP (RNA Immunoprecipitation) and RNA pull-down are key tools for our in-depth study of the interaction between RNA and protein. RIP uses specific antibodies to the target protein to isolate and purify the RNA interacting with the protein, and RNA pull-down captures the interacting protein through RNA probes. We learned about RIP technology in detail above. Today we will take you to understand RNA pull-down technology.
RNA pull-down
RNA pull-down is a technique to study the binding of intracellular RNA and protein/RNA. First, the RNA is labeled (such as biotin labeling), and then incubated with cell lysate to form an RNA-RNA/protein complex, and then the bound RNA or protein is detected. After complex elution, the interaction of target RNN with certain RNA molecules was identified by fluorescent quantitative PCR (RNA pull down-QPCR) or high-throughput sequencing (RNA pull down-seq) method, and the interaction of target RNN with certain proteins was detected by Western blot (pull down-WB) experiment and mass spectrometry (pull down-MS) technology.
RNA Pull down Technical Principles
RNA pull down The general flow of the experiment:
1. Probe preparation
Specific primers containing the T7 promoter were designed and synthesized.
PCR amplification was performed using a plasmid containing the gene of interest as a template.
The PCR product was used as a template for in vitro transcription to obtain the target RNA.
The RNA of interest is labeled, typically using biotin labeling.
2. Preparation of cell lysate
Cells were collected and lysed to prepare a cell lysate.
Cell debris was removed by centrifugation and the supernatant was collected.
3. RNA-protein complex formation
The labeled RNA probe is mixed with the cell lysate and incubated under suitable conditions so that the RNA binds to the potential protein and forms a complex.
4. Magnetic bead combination and washing
Streptavidin magnetic beads are added, which are capable of binding to biotin-labeled RNA.
Mix upside down so that the magnetic beads are fully contacted and bound to the RNA-protein complex.
The magnetic beads are separated by a magnetic field, removing unbound material.
The magnetic beads were washed multiple times to remove non-specifically bound proteins.
5. Elution of enriched proteins
Proteins bound to RNA are eluted using an appropriate elution buffer.
6. Protein detection and identification
SDS-PAGE electrophoresis and silver staining were used to preliminarily detect the enriched proteins.
Techniques such as mass spectrometry or Western blotting further identify the enriched proteins.
7. Data analysis
The mass spectrometry data was analyzed to determine the kinds of proteins interacting with the RNA.
Functional annotation and network analysis were performed on the identified proteins to understand the biological significance of RNA-protein interactions.
Construction of RNA overexpression plasmid → in vitro transcription template → RNA pull down → silver staining quality inspection → mass spectrometry identification
RNA pull down Experimental procedure
RNA pull down Frequently asked questions
1. What should we pay attention to throughout the experiment?
All reagents and consumables used in the experiment need to be RNase-removed, and the samples also need to be added with protease and RNase inhibitors, preferably sonicated. The whole process of protein lysis should be operated on ice as much as possible.
2. Besides in vitro transcription, is there any other way to obtain the target RNA?
In vitro transcription is more pure than chemical synthesis. In pull-down experiments, the target RNA is generally obtained by in vitro transcription. However, when the target RNA sequence is greater than 2000bp, it is not easy to transcribe successfully in vitro. In this case, we can design and synthesize a small piece of target RNA probe, bind to the target RNA through the probe, and then bind to the protein.
3. What is the in vitro transcription concentration of RNA before it can be used? Its OD is generally not high. Can it be used? How to quantify it?
The concentration of RNA obtained by in vitro transcription can reach more than 2μg/uL. If the OD value is not high, RNA purification can be performed. Even if it is not purified, some more RNA can be added in the experiment. Generally, agarose gel electrophoresis detection is performed for quantification, and the concentration of RNA can be determined based on the marker concentration. The concentration of RNA can also be measured with an instrument. And pull-down experiments do not require particularly accurate quantification, and generally excessive probes are added.
4. What is the difference between lncRNA primer design and ordinary primer design?
Primers for in vitro transcription amplification only need to add the T7 promoter sequence at the 5 'end of the forward primer.
5. When doing lncRNApull down + mass spectrometry, you will generally find multiple interacting proteins, right? How to choose which protein to continue research?
The number of different RNA-binding proteins is different, so it cannot be generalized. The screening proteins can be determined according to your own research direction or related functions.
Gel migration or electrophoretic mobility (EMSA)
In addition to RIP and RNA pull Down, gel migration or Electrophoretic Mobility Shift Assay (EMSA), also known as gel arrest assay or DNA binding assay, is a research technique widely used in molecular biology. It can also be used to study the interaction between proteins (especially transcription factors) and DNA or RNA.
Fundamentals
The basic principle of EMSA is to use gel electrophoresis technology to observe changes in the migration rate of proteins after they form complexes with DNA or RNA. When the protein binds to the labeled DNA or RNA fragment, the complex formed is larger than the unbound probe molecule, so it migrates slowly during electrophoresis. By comparing the ratio of free probes to bound probes in the sample, the binding of proteins to nucleic acids can be inferred.
EMSA fundamentals
Experimental procedure
Prepare the sample: Extract the protein or nuclear protein within the cell.
Labeled probes: A specific DNA or RNA sequence is labeled using radioactive or non-radioactive labeling techniques.
Protein-nucleic acid binding: The labeled probe is mixed with the protein to be tested, allowing them to bind.
Gel electrophoresis: The mixture was subjected to non-denaturing polyacrylamide gel electrophoresis.
Detection: The bands are visualized by autoradiography or other detection methods such as chemiluminescence.
EMSA-related FAQ
1. How to choose the three experimental techniques?
EMSA is mainly used to study the direct binding between RNA and protein, and is more inclined to qualitative analysis. It usually studies the corresponding protein for known RNA and is suitable for preliminary screening.
RIP is more suitable for studying the interaction of specific RNA-binding proteins with RNA under in vivo conditions.
RNA pull-down is an efficient method for directly identifying proteins that bind to specific RNAs and is suitable for in vitro studies.
Which method you choose depends on your research objectives and available resources. For example, if you want to identify which RNA molecules a specific RNA-binding protein interacts with, then RIP may be a better choice; If you want to study which proteins a specific RNA sequence interacts with, RNA pull-down may be a more appropriate choice.
2. Why can't I see the migration zone?
The protein content in the sample is insufficient; The labeled probe concentration is too low; Low labeling efficiency; Protein inactivation, ensure that the protein is not denatured or inactivated during processing, avoid repeated freezing and thawing, use freshly prepared protein samples. Insufficient exposure or detection time; Complex instability and other factors.
3. Why is the experimental background high?
The quality of protein is not high and there are many impurities; Exposure or imaging time is too long; Insufficient sealing time and poor washing effect; The membrane was not always wet during the experiment; Factors such as excessive concentration of labeled probes.
Related good things recommendations
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Item number |
Product Name |
Specifications |
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RNA Pull Down Kit |
6T |
|
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RNA co-immunoprecipitation (RIP) kit |
6T |
|
|
Silver staining kit |
20T |
|
|
Trizol |
100mL/500mL |
|
|
Immuno (co) precipitation (IP/CoIP) kit |
50T |
|
|
Chromatin co-immunoprecipitation (ChIP) kit |
22T |
|
|
DNA Pull Down Kit(Animal) |
6T |
|
|
ECL Chemiluminescence Assay Kit |
25mL×2 |
Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.
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Absin Bioscience Inc. Email: worldwide@absin.cn |
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