miRNA Immunoprecipitation Service

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miRNA Immunoprecipitation Service

As one of the world-leading biotechnology companies specializing in RNA research, IntegrateRNA has recently introduced an optimized miRNA immunoprecipitation assay to isolate and identify endogenous miRNAs, mRNAs and RBPs in mRNA complexes by immunochemistry. With rich experience in molecular biological experiments, our experts will offer you an optimal solution to facilitate your mechanism research and clinical application of miRNA.

miRNAs are incorporated into the RNA-induced signaling complex (RISC) and control the translation/degradation of their complementary mRNAs. miRNAs have recently been shown to play an important role in cell growth, development, differentiation and apoptosis. Changes in the expression of miRNAs are associated with various diseases, including cancer. It is important to identify the target mRNAs for understanding the disease mechanism.

RIP (RNA immunoprecipitation) is an antibody-based technology used to localize RNA-protein interactions in vivo. The RNA-binding protein (RBP) of interest is immunoprecipitated along with its bound RNA to identify binding RNAs (mRNA, non-coding RNA or viral RNA), which will be detected by real-time PCR, microarray or sequencing.

miRNA Immunoprecipitation AssayFigure1. The principle of miRNA immunoprecipitation

Based on your requirements, we offer two different miRNA immunoprecipitation methods:

  • RIP through RISC components (typically AGO)

The Argonaute family is a core component of RISC, in which AGO binds to miRNA and siRNA. By the immunopurification of anti-AGO antibodies, we can isolate AGO-containing RNP, which includes miRNAs, mRNAs and RBPs.

  • RIP through RBPs

We utilize RBP specific antibodies to immunoprecipitate mRNAs and their regulatory miRNAs from RBP-RNA complex. Then cluster mRNAs and miRNAs could be isolated for subsequent analysis by microarray, sequencing or RT-PCR.

Workflow of miRNA Immunoprecipitation Service

  • Preparation of cell/tissue lysates
  • Immunoprecipitation of the RBP/AGO together with the bound RNA
  • RNA isolation

miRNA Immunoprecipitation AssayFigure2. RNA isolation methods

Any of these separation methods recover miRNA more efficiently than conventional phenol extraction methods and can be used to identify disease or function-related miRNAs and to extensively analyze their target mRNAs.

  • Analysis of target RNAs (qRT-PCR, microarray and sequencing)

Technical Considerations

  • RNase pollution

We strictly monitor to ensure that the project does not contain RNase during the process.

  • Controls

Throughout the experiment, we will maintain one or more negative controls, such as no antibody samples or immunoprecipitation from knockout cells or tissues, depending on your needs. Knockdown cells are not recommended for negative control experiments.

  • Downstream analysis

Based on your project needs, we chose the best method to analyze the RNAs isolated from the IP products and use a variety of methods to confirm the results. Alternative analytical methods include sequencing, microarray, qRT-PCR.

Applications

  • Identify novel miRNAs and construct miRNA libraries
  • Identify miRNAs bound to their mRNA targets
  • Analyze the maturation mechanism of miRNA
  • Analyze intact RBP-mRNA complexes
  • Identify RISC components and analyze their function

With many years of experience in RNA research, IntegrateRNA is dedicated to providing our clients comprehensive products and services for academic research, biotechnology research, and drug discovery, including various types of RNA discovery, analysis, and related bioinformatics services. Our experienced genomics experts are able to help you solve the problems you encounter during your miRNAs researches. We guarantee the most reliable and effective research services to meet your needs. If you have any questions, please feel free to contact us.

Reference:

  1. Miriam Gagliardi. et al. RIP: RNA immunoprecipitation. Methods in Molecular Biology, 2016
For research use only. Not intended for any clinical use.
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