RIP-qPCR Service for Targeted RNA–Protein Interaction Validation

How Does RIP-qPCR Work?

RIP-qPCR follows a controlled workflow that preserves native RNA–protein complexes and produces reliable fold-enrichment values.

• Cell or tissue lysis

  Cells or tissues are gently lysed in RNase-free buffer to release intact RNA–protein complexes while limiting RNA fragmentation.

• RNA immunoprecipitation (RIP)

  A specific antibody against the target RNA-binding protein or RNA modification is added with Protein A/G beads. An IgG control is processed in parallel to capture non-specific background.

• Protein digestion and RNA purification

  After washing away unbound components, proteins are digested and co-precipitated RNA is purified using column- or reagent-based methods under RNase-free conditions.

• cDNA synthesis

  RNA from RIP, IgG, and input fractions is converted into cDNA by reverse transcription using standardised protocols.

• Quantitative RT-qPCR

  qPCR assays for each candidate RNA are run across all fractions. The resulting Ct values are used to calculate relative enrichment and fold changes within and between conditions.

This stepwise RIP-qPCR protocol is optimized specifically for RNA–protein interaction analysis, helping to minimise non-specific binding and RNA degradation. In practice, this yields consistent enrichment patterns that can be compared across replicates, time points, and experimental groups.

1. Project Consultation and Assay Design

We start with a short technical discussion to align the experiment with your goals. Together we define the RNA-binding protein or modification, select target and control transcripts, and agree on species, sample types, and biological replicates.

2. Sample Receipt and Initial QC

When your samples arrive, we log and verify them, checking identity, amount, and storage conditions. If needed, we perform small pilot checks to confirm that the material is suitable for RIP-qPCR before proceeding.

3. RIP Experiment (Lysis and Immunoprecipitation)

We perform gentle lysis and RNA immunoprecipitation using optimized buffers, commercial RIP reagents, and your chosen antibody, with IgG and input processed in parallel. The focus is to preserve native RNA–protein complexes while keeping background low and conditions consistent across all samples.

4. RNA Purification and cDNA Preparation

After immunoprecipitation, we purify RNA from RIP, IgG, and input fractions and convert it into cDNA using standardised reverse transcription protocols. This provides clean, comparable templates for downstream qPCR.

5. Quantitative RT-qPCR

We run qPCR assays for your selected targets and controls with technical replicates across all fractions. The resulting Ct values form the quantitative backbone for calculating enrichment and comparing conditions.

6. Data Analysis and Reporting

Finally, we transform raw qPCR outputs into interpretable RIP-qPCR results. You receive Ct tables, fold-enrichment summaries, and key plots, along with concise methods and QC notes, plus the option to discuss the data with our scientists for interpretation and next-step planning.

Note: Detailed protocol-level steps (lysis, IP, purification, cDNA, qPCR) are described conceptually in the earlier "How Does RIP-qPCR Work?" section; here the emphasis is on how CD Genomics organises and manages the service around your project.