Skip to main content

Ago2 Immunoprecipitation for RISC-siRNA Quantitation

 Ago2 (Argonaute 2) immunoprecipitation (IP) is a technique used to isolate RNA-induced silencing complexes (RISC) from cell lysates. This method allows for the specific enrichment of active RISC complexes bound to small interfering RNA (siRNA) or microRNA (miRNA) within cells. By isolating these complexes, researchers can then quantify the siRNA associated with Ago2, which is an essential step in determining the efficacy of RISC loading and siRNA activity.

Here’s a detailed overview of how Ago2 immunoprecipitation is performed for RISC-siRNA quantitation:

Steps in Ago2 Immunoprecipitation for RISC-siRNA Quantitation

  1. Cell Lysis and Preparation of Lysate:

    • Sample Preparation: Collect cells that have been treated with siRNA, then wash them with cold phosphate-buffered saline (PBS) to remove extracellular contaminants.
    • Lysis: Lyse the cells in a gentle, RNA-preserving lysis buffer that typically includes detergents (e.g., NP-40 or Triton X-100), protease inhibitors, and RNase inhibitors to protect both protein and RNA from degradation.
    • Centrifugation: Centrifuge the lysate to remove cell debris and keep the supernatant containing Ago2-RISC complexes.

  2. Immunoprecipitation with Ago2 Antibody:

    • Antibody Incubation: Add an anti-Ago2 antibody to the lysate and incubate under gentle rotation at 4°C for a few hours to overnight, allowing the antibody to bind to Ago2-RISC complexes.
    • Beads Addition: Use protein A/G magnetic beads or agarose beads pre-blocked with BSA or another protein to minimize non-specific binding. Add the beads to the lysate, which allows the Ago2-antibody complex to bind to the beads.
    • Washing Steps: Perform multiple washes with a low-salt buffer to remove non-specific binding while retaining Ago2-bound siRNA on the beads. Washing steps should be thorough but gentle to avoid disrupting the RISC complexes.

  3. Elution of RISC Complexes:

    • Elution from Beads: Elute the Ago2-RISC complexes from the beads. This can be achieved either by using a mild elution buffer or, in some cases, by denaturing the sample if downstream RNA quantification is not affected by protein denaturation.
    • RNA Extraction (Optional): If necessary, extract the RNA from the immunoprecipitated sample to obtain a purified siRNA fraction, often using a commercial RNA extraction kit.

  4. Quantification of siRNA:

    • qPCR or RT-qPCR: To quantify siRNA, reverse transcribe the isolated siRNA (if necessary) and perform quantitative PCR (qPCR) or reverse transcription-qPCR (RT-qPCR) to detect and quantify the siRNA specifically associated with Ago2.
    • Normalization: Normalize the siRNA levels against housekeeping controls or total input to ensure accurate quantitation of RISC-loaded siRNA. Alternatively, standard curves can be used for absolute quantification.
    • miRNA as Endogenous Controls: If applicable, endogenous miRNAs associated with Ago2 can serve as controls to assess the efficiency of the immunoprecipitation.

Considerations for Accurate RISC-siRNA Quantification

  • Antibody Specificity: Use a high-specificity anti-Ago2 antibody to minimize cross-reactivity with other proteins, as this improves the accuracy of siRNA quantitation.
  • RNase-Free Conditions: As siRNA is highly susceptible to degradation, use RNase-free reagents, pipette tips, and tubes throughout the experiment.
  • Avoiding Over-Washing: Excessive washing can lead to loss of weakly bound siRNA from Ago2, potentially underestimating siRNA levels.
  • Standard Curve for siRNA Quantification: Using a standard curve with known concentrations of the siRNA allows absolute quantification, which is useful when comparing RISC loading across different samples.

Advantages of Ago2 Immunoprecipitation for RISC-siRNA Quantification

  1. Specificity for Active RISC: This method specifically isolates siRNA bound to Ago2, allowing accurate assessment of active RISC-bound siRNA, which directly correlates with gene silencing efficacy.
  2. Insight into siRNA Efficiency: Quantifying Ago2-bound siRNA can provide insights into the efficiency of RISC loading, helping to optimize siRNA design and dosage for therapeutic applications.
  3. Application to miRNA Research: The method can also be applied to study endogenous miRNAs bound to Ago2, providing insights into both endogenous and exogenous RNA silencing mechanisms.

Limitations and Challenges

  1. Requirement for High-Quality Antibodies: The success of this assay heavily depends on the availability of high-quality, highly specific anti-Ago2 antibodies.
  2. Potential Loss of Weakly Bound siRNA: Weakly bound siRNA might dissociate during washing, potentially underestimating the true RISC-associated siRNA levels.
  3. High Sensitivity of siRNA to Degradation: Since siRNA is easily degraded, rigorous RNase-free techniques are essential, adding complexity to the workflow.

Summary

Ago2 immunoprecipitation is a valuable method for quantifying siRNA within active RISC complexes, allowing researchers to measure the extent of siRNA incorporation into RISC and evaluate its gene-silencing potential. By providing specific enrichment of Ago2-bound siRNA, this technique offers detailed insights into siRNA activity and efficacy, crucial for optimizing siRNA-based therapeutics and understanding RNA interference mechanisms.

Popular posts from this blog

Human Genome Editing: FDA Draft Guidance Summary

Consideration for Developing Gene Editing Product  1. Genome Editing Methods: Genome editing can be achieved through nuclease-dependent or nuclease-independent methods. Nuclease-dependent methods involve introducing site-specific breaks in DNA using technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), modified-homing endonucleases, and CRISPR-associated (Cas) nucleases. These breaks can lead to modification of the DNA sequence at the cleavage site. Nuclease-independent methods can change DNA sequences without cleaving the DNA and include techniques like base editing and synthetic triplex-forming peptide nucleic acids. The choice of GE technology should consider factors such as the mechanism of action, the ability to target specific DNA sequences, and the potential to optimize components for efficiency, specificity, or stability. 2. Type and Degree of Genomic Modification: Different GE approaches rely on DNA repair pathways such as ho
Read more

FDA Guidance on Studying Multiple Versions of Cellular or Gene Therapy Products in Early-Phase Clinical Trials

 The purpose of this guidance is to offer advice to sponsors interested in conducting early-phase clinical trials for a single disease involving multiple variations of a cellular or gene therapy product. Sponsors aim to gather preliminary safety and efficacy data for these product variations within a single clinical trial. It's important to note that even though multiple product versions are studied together, each version is distinct and typically requires a separate investigational new drug application (IND) submission to the FDA. The primary goal of these early-phase clinical studies is to inform decisions about which product version(s) should be advanced for further development in later-phase trials. As such, these studies are not designed to provide the main evidence of effectiveness needed for a marketing application. They are generally not statistically powered to demonstrate a significant difference in efficacy between the different study arms. In this guidance, the FDA prov
Read more

Stem loop RT-PCR for Detection of siRNA in Animal Tissues

Step Loop RT-PCR for Detection of Small Interfering RNA (siRNA) The recent publications described a novel used the novel method for the detection of siRNAs using a TaqMan®-based approach. This approach utilizes similar strategy that has been used for microRNA detection. The approach is illustrated in below.  In brief, the RT step occurs in the presence of a stem-loop RT primer that is complementary to the last 6–10 bases of the 3′ end of the antisense strand of the target siRNA. The stem-loop primer contains an additional universal sequence at the 5′ end that facilitates a TaqMan-based detection strategy in the subsequent qPCR step. As in the case of microRNA, the forward primer for qPCR is sequence-specific for the target siRNA. For sequence compositions that yield a low predicted melting temperature (Tm), the forward primer is designed as a tailed primer to help increase Tm. Stem Loop PCR for SiRNA Detection Step 1: Preparation of liver and plasma samples for the quantification of si
Read more

Human Gene Therapy for Neurodegenerative Diseases: FDA Guidance Summary

  Neurodegenerative diseases are a diverse group of disorders characterized by the progressive degeneration of the central or peripheral nervous system, and they can have various causes and clinical characteristics. This guidance document is a resource for sponsors on different aspects of product development, preclinical testing, and clinical trial design. It acknowledges the unique challenges and considerations associated with developing GT products for such complex and varied diseases. Below are the key summaries from the guidance. CONSIDERATIONS FOR CHEMISTRY, MANUFACTURING AND CONTROLS (CMC) The considerations for Chemistry, Manufacturing, and Controls (CMC) when developing gene therapy (GT) products for the treatment of neurodegenerative diseases are crucial for ensuring the safety and efficacy of these advanced therapies. Here, we will elaborate on the specific CMC considerations outlined in your text: Route of Administration and Product Volume: Neurodegenerative diseases often r
Read more

Standard Template For Clinical Study Report (CSR)-Gene Therapy

 A Standard Format for a Clinical Study Report (CSR) typically includes the following sections and components: Title Page: Title of the Clinical Study Report Study Title Protocol Number Version Date Sponsor's Name and Logo Date of Report Compilation Table of Contents: A list of all sections, subsections, and appendices with page numbers for easy navigation. List of Abbreviations and Glossary: A compilation of all abbreviations used throughout the report, along with their definitions. Executive Summary: A concise overview of the study, including objectives, methods, key findings, and conclusions. Introduction: Background and rationale for the study. Study objectives and hypotheses. Study Design and Methods: Detailed information about the study design, including: Inclusion and exclusion criteria. Study population and recruitment. Randomization and blinding procedures. Data collection methods and tools. Statistical analysis plan. Ethical Considerations: Information on ethical approval
Read more