RNA Immunoprecipitation Sequencing (RIP-Seq)

RNA immunoprecipitation sequencing (RIP-seq) is a next-generation sequencing (NGS)-based method to comprehensively study the situation of intracellular RNA and its binding protein, a dynamic process of the post-transcriptional regulatory network. We provide a one-stop RIP-seq service to help customers discover new RNA-protein interaction sites and explore more gene regulation functions.

Overview

Currently, except for a few RNAs that can function alone in the form of ribozymes, most RNAs are combined with proteins to form RNA-protein complexes. RNA-binding proteins (RBPs) play an important role in the process of gene regulation such as RNA synthesis, alternative splicing, modification, transport, and translation. Therefore, studying the interaction of RNA and protein is the key to exploring RNA functions. RNA immunoprecipitation (RIP) is an important technique for studying the interaction of protein and RNA in vivo. The technology uses antibodies against the target protein to precipitate the corresponding RNA-protein complex, and analyze RNA after separation and purification. The advent of sequencing technologies, coupled to various RIP chemistries, has enabled the simultaneous detection of thousands of bound transcripts in a single experiment. Based on RIP, RIP-seq uses specific antibodies to immunoprecipitate RNA-binding proteins or specially modified RNAs. After RNA isolation, Illumina sequencing is used to study RNA regions or types specifically bound by specific proteins across the full transcriptome, and differences between multiple samples can be compared. Our company provides RIP-seq to obtain insights into not just the well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs. RIP-Seq is a powerful tool for understanding the dynamic process of post-transcriptional regulatory networks. It provides researchers with the possibility to better understand the mechanisms of post-transcriptional regulation and even gene expression in vivo.

Workflow of RIP Sequencing Service – CD Genomics

Features

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Raw data quality control
• Quality assessment of sequencing data
• Peak calling and visualization
• Peak identification
• Peak distribution
• Peaks annotation
• Motif search of enrichment sites
• Differential binding analysis
• Alterative splicing analysis
• GO and KEGG pathway analysis
• Clustering analysis and enrichment analysis

Sample Requirements

RNA sample quantity ≥ 50 ug.
OD260/280 ≥ 1.8, OD260/230 ≥ 1.5.
Please make sure that the RNA is not significantly degraded.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

Case Studies

• • Unveiling the Role of SORBS2 in Ovarian Cancer Metastasis through RIP-seq & RNA-seq

    • Background

      Ovarian cancer stands as one of the most lethal gynecological malignancies affecting women worldwide. With limited effective early detection methods and treatment modalities, the rates of late-stage diagnosis and disease recurrence remain alarmingly high. The research project aimed to shed light on the role of SORBS2, an RNA-binding protein, in curbing the metastatic potential of ovarian cancer. The authors recognized the urgent need for innovative therapeutic strategies that target key processes in ovarian cancer development, particularly metastasis, and thus chose to explore the potential of RBPs as a novel avenue for intervention.

      Methodology

      The researchers utilized two cutting-edge techniques, RIP-Seq and RNA-seq, to comprehensively investigate the role of SORBS2 and its associated molecular pathways in ovarian cancer metastasis.

      RIP-Seq, or RNA immunoprecipitation sequencing, was employed to identify RNA transcripts that specifically bind to the SORBS2 protein. By isolating SORBS2-associated RNAs, the researchers gained insight into the specific molecular targets of SORBS2's regulatory activity. This technique allowed them to focus their subsequent analyses on RNAs that interact with SORBS2, potentially influencing its ability to inhibit ovarian cancer metastasis.

      SORBS2 depletion affects the stability of transcripts directly bound by SORBS2. (Zhao et al., 2018)

      In order to further understand the downstream effects of SORBS2 and identify potential metastasis-related targets, the researchers performed RNA-seq analysis. This technique enabled them to profile the global gene expression changes that occurred in response to SORBS2 knockdown. By comparing the transcriptomes of cells with reduced SORBS2 expression to control cells, the researchers were able to identify transcripts whose stability was affected by SORBS2 depletion.

      Results and Findings

      The combination of RIP-Seq and RNA-seq analyses yielded intriguing insights into the role of SORBS2 in ovarian cancer metastasis. The researchers identified specific RNA transcripts that directly bound to SORBS2 through RIP-Seq. Subsequent RNA-seq analysis revealed that knockdown of SORBS2 led to reduced stability of transcripts associated with genes WFDC1 and IL-17D. Importantly, functional validation experiments confirmed that both WFDC1 and IL-17D played a role in inhibiting the metastatic colonization of ovarian cancer cells.

• • Role of SUMO1 Modification on KHSRP in Regulating Tumorigenesisthrough RIP-seq & miRNA-seq

    • Background

      MicroRNAs (miRNAs) play a crucial role in the regulation of various physiological and pathological processes, including cancer. Small ubiquitin-like modifier (SUMO) proteins are known to modulate protein function and localization through post-translational modification. Recent research has uncovered the involvement of SUMOylation in the regulation of the miRNA pathway. Additionally, KHSRP, a single-stranded nucleic acid binding protein, is recognized for its roles in transcription, mRNA decay, and miRNA biogenesis. KHSRP is part of the Drosha-DGCR8 complex, essential for miRNA maturation.

      Methods

      To investigate the relationship between SUMOylation and KHSRP in the context of tumorigenesis, a comprehensive array of methods was employed:

      • In Vivo SUMOylation Assay: Ni2+-NTA affinity pulldown and immunoprecipitation (IP) techniques were utilized to analyze the SUMOylation status of KHSRP within living cells.
      • miRNA Biogenesis Study: High-throughput miRNA sequencing, quantitative RT-PCR, and RNA immunoprecipitation (RIP) assays were performed to assess how KHSRP's SUMO1 modification affects miRNA biogenesis.

      SUMOylation of KHSRP at K87 inhibits the biogenesis of miRNAs. (Yuan et al., 2017)

      Results

      The study revealed significant insights into the relationship between KHSRP, SUMO1 modification, and tumorigenesis:

      • KHSRP SUMOylation: The researchers discovered that KHSRP undergoes SUMO1 modification at a key site, K87. This modification is influenced by the tumor microenvironment, with hypoxia increasing SUMOylation while growth factors decrease it.
      • Localization Changes: SUMO1 modification was found to impact the cellular distribution of KHSRP, potentially promoting its translocation from the nucleus to the cytoplasm.
      • Effects on miRNA Biogenesis: SUMO1 modification hindered the interaction between KHSRP and the pri-miRNA/Drosha-DGCR8 complex, disrupting the processing of pre-miRNAs from pri-miRNAs. This disruption particularly affected miRNAs with short G-rich stretches in their terminal loops (TL). As a consequence, the downregulation of specific TL-G-rich miRNAs, including the let-7 family, was observed.
      • Tumorigenesis Implication: The altered miRNA biogenesis due to SUMO1 modification of KHSRP correlated with tumorigenesis. Functional assays demonstrated that this modification led to enhanced tumor cell behaviors in vitro, while the xenograft tumor model in mice provided in vivo validation of the role of KHSRP SUMOylation in driving tumorigenesis.

FAQ

• • Can extracted RNA be used for RIP experiments?

    • No, extracted RNA is not suitable for RIP experiments. RIP-Seq relies on the use of a specific antibody to selectively capture RNA-binding proteins (RBPs) bound to RNA sequences. Since the goal is to measure the interaction between RBPs and RNA, there should be no RBPs left in the extracted total RNA. Attempting RIP with extracted RNA would not yield meaningful results as it lacks the essential protein-RNA complexes required for the experiment.

• • I want to perform a RIP (RNA Immunoprecipitation) experiment. What information should I provide and confirm?
    • • Sample Type: Describe your sample, whether it's cells, tissues, or fluids.
      • RBP of Interest: Specify the RNA-binding protein you're studying.
      • Availability of IP Antibody: Confirm the availability of a validated IP antibody for your RBP.
      • RNA Sequence Target: Identify the RNA sequences your RBP binds (e.g., mRNA, miRNA, lncRNA, circRNA).
      • Other Relevant Details: Include any pertinent experimental context or objectives.
• • Should I Conduct a Western Blot Pre-Experiment Before RIP?

    • Yes, a Western Blot pre-experiment is highly recommended before starting RNA Immunoprecipitation (RIP) for the following reasons:

      • Verify Antibody Specificity
        Ensure that the antibody binds specifically to your target protein.
        Confirm that it does not cross-react with other proteins.
      • Confirm Target Protein Presence
        Check if the target protein is present in your sample.
        Determine its abundance level, which is crucial for RIP success.

      Performing this pre-experiment will improve the reliability of your RIP results by ensuring the quality of your antibody and the suitability of your sample.

• • Why do I see bands for the target protein in Western blotting (WB), but not in RNA immunoprecipitation (RIP)?

    • The presence of bands in Western blotting (WB) but not in RNA immunoprecipitation (RIP) could be due to the differing requirements of these techniques. While your current antibody works well for WB, it may not be suitable for RIP. In RIP experiments, it's crucial to use antibodies designated as "IP grade" to ensure optimal results. These IP grade antibodies are specifically designed and validated for immunoprecipitation, making them the recommended choice for RIP assays.

• • What controls are used in RIP sequencing?

    • RIP sequencing relies on critical controls to ensure data accuracy and reliability. These controls serve key purposes:

      • Input RNA Control: Identifies initial RNA composition before immunoprecipitation.
      • Mock IP Control: Differentiates true interactions from background noise.
      • Chromatin Break Validation: Confirms RNA integrity during chromatin fragmentation.
      • IP Validation Control: Verifies efficient target RNA-protein complex isolation.

      These controls enhance the precision of RIP sequencing results, facilitating robust insights into RNA-protein interactions.

References:

1. Zhao, Linjie, et al. "The RNA binding protein SORBS2 suppresses metastatic colonization of ovarian cancer by stabilizing tumor-suppressive immunomodulatory transcripts." Genome Biology 19 (2018): 1-20.
2. Yuan, Haihua, et al. "SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis." Molecular Cancer 16.1 (2017): 1-18.