Circular RNAs (circRNAs) function as miRNA sponges or protein decoys, but confirming direct molecular interactions is essential for mechanistic studies. Our combined pull-down and firefly/renilla luciferase assay service provides orthogonal validation of circRNA–miRNA and circRNA–RBP binding.
The circRNA pull-down assay uses a biotinylated probe complementary to the back-splice junction to capture interacting molecules from cell lysate, followed by streptavidin bead enrichment and qRT-PCR or Western blot detection. A parallel firefly/renilla luciferase reporter assay confirms functional regulation by cloning the circRNA target sequence downstream of a luciferase gene and measuring changes in reporter activity upon co-transfection with miRNA mimics or RBP expression plasmids.
This integrated approach is widely applied in studies of circRNA mechanisms in cardiovascular diseases, cancer, and developmental biology, where high-confidence evidence of interaction is required for publication and downstream functional work.
Key Features
Biotinylated RNA Probe Design and Synthesis
Probes are designed against the back-splice junction of your circRNA of interest, labeled with biotin at the 3′ or 5′ end, and quality-checked by HPLC or PAGE to ensure correct size and integrity. Multiple probes can be designed for isoforms or long circRNAs as needed.
Integrate physical binding evidence (pull-down) with functional evidence (luciferase) within a single project. This two-assay workflow delivers coherent datasets that satisfy journal reviewers and streamline mechanistic conclusions.
Custom Reporter Construct Cloning
Wild-type and mutant circRNA target sequences are cloned into firefly/renilla luciferase reporter vectors downstream of the luciferase gene, enabling precise comparison of binding-dependent regulation for each candidate interaction.
Multi-Modal Detection Readouts
Choose from qRT-PCR, Western blot, small-RNA sequencing, or mass spectrometry to detect pulled-down miRNAs and proteins. Each readout is matched to your research question and target abundance.
Comprehensive Built-In Controls
Every workflow includes RNase-treated, mutant-probe, no-probe, and empty-vector controls. These mandatory controls minimize false positives and produce publication-ready, defensible datasets.
Assay Modalities
Our service covers a range of interaction types and readout options, customized per project scope. Each modality can be run independently or combined for orthogonal validation.
CircRNA–miRNA Pull-Down: Biotin-labeled full-length or junction-spanning probe captures miRNA bound to circRNA from lysate or synthetic RNA. Bound miRNA is quantified by qRT-PCR using miRNA-specific primers or by small RNA sequencing for discovery.
CircRNA–RBP Pull-Down: Probe incubations with nuclear or cytoplasmic extracts to pull down RNA-binding proteins. Eluted proteins are identified by Western blot (targeted) or mass spectrometry (global).
Firefly/Renilla Luciferase Reporter (circRNA–miRNA): Reporter vector containing the circRNA target site; co-transfection with miRNA mimic/inhibitor to measure luciferase suppression or derepression.
Firefly/Renilla Luciferase Reporter (circRNA–RBP): RBP overexpression or knockdown plasmids co-transfected with the reporter to assess modulation of translation or RNA stability.
Multiplex Detection Options: Simultaneous pull-down of multiple circRNA targets in one reaction (if probes have distinguishable labels) or parallel firefly/renilla luciferase constructs for multiple candidate interactions.
Workflow
Our integrated workflow combines probe-based pull-down and reporter-based luciferase assays, with each step optimized for specificity, reproducibility, and traceability.
Consultation and Target Submission: You provide circRNA sequence (full-length or junction), candidate miRNA/RBP names, and cell type. We confirm feasibility, design probes and firefly/renilla luciferase constructs, and finalize the experimental plan.
Probe and Reporter Construction: Biotinylated RNA probes are synthesized and quality-checked. Firefly/renilla luciferase reporter plasmids are constructed containing the wild-type or mutant circRNA target region.
Pull-Down Assay and Validation: Probes are incubated with cell lysate or synthetic interactors, captured on streptavidin beads, washed, and eluted. RNase A treatment and mutant probe controls are run in parallel. Bound RNAs/proteins are detected by qRT-PCR or Western blot.
Firefly/Renilla Luciferase Reporter Assay: Reporter plasmids are co-transfected into the specified cell line with miRNA mimics/inhibitors or RBP expression plasmids. Luciferase activity is measured 24–48 h post-transfection, normalized to Renilla, and fold-change calculated.
Data Analysis and Reporting: Raw data are normalized, statistically analyzed (≥3 biological replicates), and presented in a final report with graphs, tables, and interpretation. Deliverables include pull-down qPCR/Western blot data and luciferase assay results.
Two Assays, One Project
By combining pull-down and firefly/renilla luciferase within a single project, you receive physically grounded and functionally validated evidence of interaction—saving time, reducing inter-batch variability, and giving you publication-ready datasets.
Project Milestones
Each project follows a structured timeline. The exact duration depends on the complexity of the assay and the number of targets. Typical milestones are outlined below.
Initial Design and Synthesis: Project kickoff, probe and reporter design, sequence confirmation, and initial synthesis of biotinylated probes and reporter constructs.
Assay Optimization: Optimization of probe hybridization, bead capture, and transfection conditions for the selected cell line.
Pull-Down Execution: Full pull-down assay with controls, followed by detection via qRT-PCR, Western blot, or sequencing.
Final Report Delivery: Comprehensive report with raw and normalized data, statistical analysis, representative figures, and a methods section suitable for manuscript inclusion.
Timelines are confirmed during project initiation based on scope and current capacity.
Quality Assurance
Each service order is executed under a documented quality system. Probe purity is verified by HPLC; biotinylation efficiency is confirmed by streptavidin gel-shift assay. Pull-down specificity is monitored through RNase-treated and mutant probe controls, and only experiments meeting pre-defined signal-to-noise ratios (≥5-fold enrichment over negative control) proceed to detection.
For firefly/renilla luciferase assays, all transfections are performed in triplicate with internal normalization (Renilla luciferase). Acceptance criteria include coefficient of variation within replicates and at least 2× change in luciferase activity for validated interactions. Raw data files, normalization calculations, and statistical tests are included in the final deliverable.
Our scientists review every dataset prior to release to ensure reproducibility and accuracy, and a senior scientist signs off on the final report.
Why Choose Our Service
Customized Probe Design: Probes are tailored to your circRNA's back-splice junction, avoiding linear host interference. Multiple probe designs are tested for optimal capture efficiency.
Integrated Two-Assay Validation: Combine pull-down (direct binding evidence) with firefly/renilla luciferase (functional evidence) in one project, saving time and ensuring coherent data sets.
Comprehensive Controls Included: Mandatory controls (RNase, mutant probe, no-probe, empty vector) are built into every workflow, reducing false positives and providing publication-ready results.
Expert Data Analysis and Reporting: Our bioinformatics and molecular biology teams prepare detailed reports with normalized values, statistical analysis, and graphical visualization, ready for manuscript inclusion.
Backed by Creative Biogene: All projects are delivered under Creative Biogene's ISO-aligned quality system, with dedicated project management, milestone-based reporting, and full intellectual property protection for your data.
Flexible Project Scope: From single-target validation to multiplex screens, our service is scoped to match the size and complexity of your study.
Supported Cell Types
We routinely handle a variety of cell types for pull-down lysate preparation and firefly/renilla luciferase transfection.
Adherent Cell Lines: HEK293, HeLa, A549, HepG2, MCF-7, and many others. Transfection and lysate preparation optimized for high yield.
Suspension Cells: Jurkat, K562, and other hematopoietic lines. Adjusted protocols for electroporation or lipofection.
Primary Cells: Cardiomyocytes, hepatocytes, pulmonary fibroblasts, and other primary cultures. Limited passage numbers accommodated with cell type-specific lysis buffers.
Induced Pluripotent Stem Cells (iPSCs): PSCs and differentiated derivatives (e.g., neurons, myocytes) are supported with feeder-free culture and optimized nucleofection conditions.
Contact Us
Whether you are validating a circRNA–miRNA sponge, mapping circRNA–RBP interactions, or building mechanistic evidence for publication, the scientific team at Creative Biogene can help you design the most appropriate pull-down and firefly/renilla luciferase strategy—from probe design and cell line choice to readout selection and data interpretation. Reach out to discuss a tailored solution for your next project.
