Native Regulatory Complexes Characterization for Transcriptome Studies
Creative Biolabs uses RNA immunoprecipitation sequencing (RIP-Seq) for purification of endogenous RNA-protein complexes under physiological conditions in native conformation. Creative Biolabs crosslinking-free RIP-Seq workflow preserves native conformation of endogenous regulatory complexes and allows determination of the steady-state native complex composition. Our Native RIP-Seq is offered for non-clinical research studies only and is ideal for mechanistic studies related to transcriptome regulation and disease biology.
RIP-Seq (RNA Immunoprecipitation Sequencing) is a method for identification of native RNA-protein interactions. It involves immunoprecipitating endogenous RNA-binding proteins (RBPs) using specific antibodies and subsequently high-throughput sequencing of the co-precipitated RNAs. In contrast to crosslinking-based approaches, RIP-Seq preserves the physiological stoichiometry of RNA-protein complexes since the interactions are not covalently stabilized by artificial crosslinks, allowing the mapping of steady-state RNA-protein interactions under near-physiological conditions. RIP-Seq is therefore particularly well-suited to study constitutive RNA-protein regulatory networks, such as RBP-housekeeping RNA interactions or functional modules of non-coding RNAs, with minimal technical biases.
| Method | RIP-Seq | CLIP-Seq | ChIRP-Seq |
|---|---|---|---|
| Complex State | Native steady-state | Crosslinked transient | Chromatin-tethered |
| Resolution | Domain-level (200-500bp) | Nucleotide-level | Chromatin proximity |
| Use | Constitutive interactomes | Splicing dynamics | Chromatin-RNA hubs |
Fig.1 RIP-Seq in RNA-Protein Interaction Analysis Service.
Native RIP-seq is an antibody-based immunoprecipitation approach to capture endogenous RNA-protein complexes in native conformation, without crosslinking artifacts. We perform native RIP-seq to map endogenous protein-RNA interactions involved in basic biological research on constitutive RBPs-RNA interactions, such as biogenesis of ribonucleoproteins and housekeeping regulatory circuits, with native interaction maps obtained from research grade cell lines. CreatiVE’s Native RIP-seq is performed under stringent conditions with minimalistic lysis buffer for immunoprecipitation and careful control of isotype IgG samples. This preserves the native stoichiometry of RNA-protein complexes, allowing the analysis of native steady-state interactomes for non-clinical mechanistic studies only.
Our ncRNA Interactome service is specifically designed and optimized for characterizing functional RNA-protein units with a special focus on the regulatory nodes composed of long non-coding RNAs (lncRNA), circular RNAs (circRNA) and microRNAs (miRNA). We preserve strand specificity by using strand-specific library prep and a small RNA optimized protocol for capturing complexes bound to non-coding RNAs, and map RNA-protein interactions involved in processes such as miRNA-mRNA regulation or phase separation driven condensates formed by ncRNA and proteins in a disease model. Directionality of interactions is also preserved for insights into post transcriptional regulatory mechanisms. We further provide comparison to background control after annotation of peaks to non-coding RNA databases to understand ncRNA-specific binding networks.
Our Dynamic Network Analysis service enables condition or time-dependent studies to track the reprogramming of RBP targets in stress response, differentiation, or upon drug treatment. We use RIP-seq (narrowly profiled IP of interest) to track these changes, which can be correlated to transcriptome changes (RIP-seq + RNA-seq) and deconvoluted by differential peak analysis to pinpoint condition specific changes such as RNA stability mediated by RBPs in response to chemotherapy or dynamic transcriptome rewiring during development or differentiation.
RIP-Seq provides a refined view of myogenic regulatory circuits by maintaining the integrity of MyoD mRNA-HuR protein complexes during the differentiation of C2C12 myoblasts. This approach reveals the sequential recruitment of RBPs to critical transcription factors such as MYOG, elucidating the RNA-protein interaction dynamics that orchestrate myocyte differentiation while avoiding the conformational alterations caused by UV crosslinking, a vital methodological benefit for developmental biology research in model organisms.
RIP-Seq can be applied to plant-pathogen interactions, such as the Arabidopsis-thaliana response to fungal pathogens, to profile the relocalization of RNA-binding proteins to specific gene transcripts in defense responses. By preserving native RNA-protein complexes, RIP-Seq can uncover how pathogen recognition leads to post-transcriptional upregulation of immune response genes, offering unique insights into RNA-level disease resistance mechanisms, with applications strictly in agricultural research.
Employ RIP-Seq to map RBP-mediated regulation of glycolytic enzyme expression in pancreatic cancer organoids, focusing on the stabilization of HK2 and LDHA mRNAs by IGF2BP1 under hypoxic conditions. This method, by circumventing crosslinking artifacts that can distort the kinetics of metabolic enzymes, will reveal genuine RNA-protein interactions that contribute to the metabolic rewiring associated with the Warburg effect, intended for non-clinical research on tumor metabolism.
Methodological Excellence
Analytical Rigor
Preclinical Integrity
Share your target RNA-binding protein (RBP), research-grade biological model, and mechanistic objectives with our specialists. We will deliver a customized proposal featuring:
[Contact Our RNA Biology Team] to schedule a technical consultation within 48 hours and advance your transcriptome regulation research.
A: RIP-Seq captures native complexes without crosslinking, while CLIP-Seq stabilizes transient interactions.
A: Sequential IP or co-IP strategies enable multiplexed analysis.
A: Standard protocols require research-grade cultured cells or tissue samples.
A: Yes, de novo motif discovery and known RBP motif matching are standard.
Reference
