RNA and Protein Extraction from Organoids: Standard Protocols and Support from ANT BIO PTE. LTD.

1. Concept

Organoids are three-dimensional (3D) in vitro culture systems derived from stem cells or tissue fragments that recapitulate the structural and functional characteristics of their corresponding in vivo organs. As powerful models for studying development, disease mechanisms, and drug responses, organoids require efficient extraction of high-quality RNA and proteins to enable downstream molecular analyses. RNA extraction from organoids aims to isolate intact, contaminant-free total RNA for applications such as RT-PCR, real-time quantitative PCR (qPCR), and cDNA library construction. Protein extraction, by contrast, involves the isolation of subcellular fraction-specific proteins (cytoplasmic, nuclear, membrane, and structural proteins) to preserve their biological activity and enable functional studies (e.g., Western blotting, enzyme activity assays, electrophoretic mobility shift assays). The success of these extractions hinges on specialized reagents and protocols tailored to the unique 3D structure of organoids, ensuring minimal degradation and maximum yield.

2. Research Frontiers

Contemporary research on organoids is rapidly advancing towards more physiologically relevant models and high-throughput applications, driving innovations in RNA and protein extraction techniques. Key frontiers include:

• Extraction from specialized organoid types (e.g., tumor organoids, brain organoids, and patient-derived organoids) with complex architectures and extracellular matrices.
• Development of rapid, scalable extraction methods compatible with high-throughput drug screening and single-cell omics analyses.
• Optimization of subcellular protein fractionation to study organelle-specific functions and protein localization in 3D contexts.
• Integration of extraction protocols with downstream technologies such as spatial transcriptomics and proteomics to map molecular heterogeneity within organoids.

These frontiers demand reagents that preserve molecular integrity while accommodating the unique challenges of 3D cultures, such as matrix (Matrigel) degradation and efficient cell lysis.

3. Research Significance

Efficient RNA and protein extraction from organoids is critical for unlocking their full potential as research tools. From a basic science perspective, high-quality RNA enables the analysis of gene expression profiles, alternative splicing, and non-coding RNA dynamics, providing insights into organ development and disease pathogenesis. Protein extraction, particularly subcellular fractionation, allows the investigation of protein-protein interactions, post-translational modifications, and signaling pathway activation in a physiologically relevant 3D context. Clinically, these extractions support the development of personalized medicine—patient-derived organoids paired with molecular analyses enable drug sensitivity testing and biomarker discovery, guiding treatment decisions for diseases such as cancer. Additionally, reliable extraction protocols ensure reproducibility across studies, facilitating the translation of preclinical findings to clinical applications.

4. Relevant Mechanisms, Extraction Protocols, and Product Applications

4.1 Core Principles of Organoid Extraction

• RNA preservation: The primary challenge is to inhibit RNase activity (ubiquitous in cells and environments) and prevent RNA degradation during lysis and purification. This requires RNase-free reagents and rapid processing at low temperatures.
• Protein integrity: Proteins are susceptible to degradation by proteases and dephosphorylation by phosphatases. Extraction protocols must include inhibitors to preserve protein structure and function, particularly for subcellular fractionation where spatial separation adds complexity.
• Matrix dissociation: Organoids are typically embedded in matrix (e.g., Matrigel), which must be gently degraded to release intact organoids without damaging cells, ensuring efficient lysis and extraction.

4.2 Standard Extraction Protocols

ANT BIO PTE. LTD. provides specialized reagents and step-by-step protocols for reliable RNA and protein extraction from organoids, tailored to preserve molecular quality and yield.

4.2.1 Organoid Protein Extraction (Subcellular Fractionation)

This protocol uses the Cytoplasmic, Nuclear, and Membrane Protein Extraction Kit (abs9346) to isolate four protein fractions (cytoplasmic, nuclear, membrane, and structural proteins) from organoids.

Reagents Provided by ANT BIO PTE. LTD.:

• Cytoplasmic, Nuclear, and Membrane Protein Extraction Kit (abs9346): Includes cytoplasmic, nuclear, membrane, and structural protein extraction buffers.
• Organoid Passage Buffer (abs9730): Facilitates matrix dissociation and gentle organoid collection.
• Phosphatase Inhibitor II: Prevents protein dephosphorylation.
• Phenylmethylsulfonyl fluoride (PMSF): Protease inhibitor to prevent protein degradation.

Protocol:

1. Reagent Preparation:
• Thaw all kit buffers at room temperature. After thawing, place all buffers except the structural protein extraction buffer on ice immediately.
• Warm the structural protein extraction buffer at 37°C until transparent, then keep at room temperature.
• Prepare working buffers fresh before use: Add Phosphatase Inhibitor II (1:50 dilution) and PMSF (1:100 dilution) to each extraction buffer (cytoplasmic, nuclear, membrane, structural) based on the sample volume.
2. Organoid Collection and Washing:
• Aspirate medium from culture wells. Add 1-2 mL of pre-chilled (4°C) Organoid Passage Buffer (abs9730) per well, gently pipette to dissociate matrix，and collect organoids into 15 mL centrifuge tubes (pool 5 wells of a 24-well plate per tube).
• Incubate at 4°C for 10 minutes or -20°C for 5 minutes to soften matrix. Centrifuge at 300g for 5 minutes at 4°C, then discard the supernatant.
• Resuspend the pellet in 1 mL of Organoid Passage Buffer (abs9730), centrifuge at 300g for 5 minutes at 4°C, and discard the supernatant.
3. Subcellular Protein Extraction:
• Cytoplasmic protein extraction: Add 2 mL of cytoplasmic protein extraction working buffer to the organoid pellet. Vortex at 4°C for 20 minutes, then pipette the solution 50-90 times with a 1-5 mL syringe. Centrifuge at 15,000g for 10 minutes at 4°C. Collect the supernatant (cytoplasmic protein) into a new tube.
• Nuclear protein extraction: Resuspend the pellet from the previous step in 4 mL of ice-cold cytoplasmic protein extraction working buffer. Vortex at 4°C for 5 minutes, centrifuge at 15,000g for 10 minutes at 4°C, and discard the supernatant. Add 1 mL of ice-cold nuclear protein extraction working buffer to the pellet, vortex at 4°C for 5 minutes, centrifuge at 15,000g for 10 minutes at 4°C. Collect the supernatant (nuclear protein).
• Membrane protein extraction: Add 1 mL of ice-cold membrane protein extraction working buffer to the pellet from nuclear extraction. Vortex at 4°C for 5 minutes, centrifuge at 15,000g for 10 minutes at 4°C. Collect the supernatant (membrane protein).
• Structural protein extraction: Add 0.5 mL of room temperature or 37°C-warmed structural protein extraction working buffer to the remaining pellet. Vortex at 4°C for 5 minutes, centrifuge at 15,000g for 10 minutes at 4°C. Collect the supernatant (structural/cytoskeletal protein).
4. Protein Storage: Store all protein fractions at -70°C. They are suitable for Western blotting, EMSA, footprinting, reporter gene assays, and enzyme activity measurements.

4.2.2 Organoid RNA Extraction

This protocol uses the Ultra-Pure Total RNA Rapid Extraction Kit (with DNase I, abs60026) to isolate high-purity, DNase I-digested RNA from organoids.

Reagents Provided by ANT BIO PTE. LTD.:

• Ultra-Pure Total RNA Rapid Extraction Kit (with DNase I, abs60026): Includes RL lysis buffer, adsorption columns (RA), collection tubes, deproteinization buffer (RE), DNase I, RDD buffer, wash buffer (RW), and RNase-free H₂O.
• Organoid Passage Buffer (abs9730): For organoid collection and matrix dissociation.

Protocol:

1. Organoid Collection and Washing: Follow steps 2.1-2.3 of the protein extraction protocol to collect and wash organoids. Transfer the final pellet to a 1.5 mL RNase-free EP tube.
2. RNA Extraction:
• Add 1 mL of RL lysis buffer (kit component) to the organoid pellet. Gently pipette to mix, ensuring complete lysis and RNase inactivation.
• Vortex vigorously and incubate at 15-30°C for 5 minutes to fully disrupt nucleoprotein complexes.
• Add 0.2 mL of chloroform per 1 mL of RL buffer. Cap tightly, vortex vigorously for 15 seconds, and incubate at room temperature for 3 minutes.
• Centrifuge at 12,000 rpm for 10 minutes at 4°C. The sample will separate into three layers: lower organic phase, middle layer, and upper aqueous phase (containing RNA). Transfer the aqueous phase (≈60% of the original RL volume) to a new RNase-free tube.
• Add an equal volume of 70% ethanol to the aqueous phase, invert to mix (precipitation may form). Transfer the mixture (including precipitation) to an RA adsorption column (inserted into a collection tube).
• Centrifuge at 12,000 rpm for 45 seconds, discard the filtrate, and reinsert the column into the collection tube.
• Add 400 μL of deproteinization buffer (RE) to the column, centrifuge at 12,000 rpm for 45 seconds, and discard the filtrate.
• Prepare DNase I working solution: Mix 10 μL of DNase I stock solution with 70 μL of RDD buffer in a new RNase-free tube.
• Add 80 μL of DNase I working solution to the center of the column membrane. Incubate at room temperature for 15 minutes (or 37°C for 15 minutes if room temperature digestion is insufficient).
• Add 400 μL of deproteinization buffer (RE) to the column, centrifuge at 12,000 rpm for 45 seconds, and discard the filtrate.
• Add 500 μL of wash buffer (RW, ensure ethanol has been added) to the column, centrifuge at 12,000 rpm for 45 seconds, and discard the filtrate. Repeat this wash step once.
• Reinsert the column into the empty collection tube, centrifuge at 12,000 rpm for 2 minutes to remove residual wash buffer (ethanol contamination inhibits downstream reactions).
• Transfer the column to a new RNase-free tube. Add 50-80 μL of pre-warmed (65°C) RNase-free H₂O to the center of the membrane. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to elute RNA. For higher yield, reapply the eluate to the column and centrifuge for 1 minute.
3. RNA Application: The purified RNA is suitable for Northern blotting, RT-PCR, real-time RT-PCR, cDNA library construction, and in vitro translation.

4.3 Product Advantages and Applications

ANT BIO PTE. LTD.’s specialized reagents are optimized to address the unique challenges of organoid extraction:

• Protein extraction kit (abs9346): Enables efficient subcellular fractionation with dedicated buffers for cytoplasmic, nuclear, membrane, and structural proteins. Inclusion of phosphatase and protease inhibitors preserves protein activity and phosphorylation status.
• RNA extraction kit (abs60026): Integrates DNase I digestion to eliminate genomic DNA contamination, ensuring high-purity RNA for sensitive downstream applications. The rapid protocol minimizes RNA degradation.
• Organoid Passage Buffer (abs9730): Gently dissociates matrix without damaging organoids, facilitating efficient collection and washing while maintaining cell integrity.
• Supporting reagents: Matrigel (Low Factor, Phenol Red-Free, abs9495) and organoid medium kits (e.g., abs9444 for liver cancer, abs9445 for colorectal cancer) provide a complete workflow from organoid culture to extraction.

5. Brand Mission

ANT BIO PTE. LTD. is dedicated to empowering organoid research through the development and supply of high-quality, innovative, and reliable life science reagents. We strive to support researchers worldwide in unlocking the potential of organoids as models for development, disease, and drug discovery. By adhering to rigorous quality control standards, optimizing protocols for 3D culture systems, and offering comprehensive product portfolios, we aim to provide one-stop solutions for organoid culture, extraction, and molecular analysis. Our mission extends beyond product provision to fostering scientific collaboration and knowledge sharing, driving breakthroughs that translate into improved human health.

6. Related Product List

7. Brand Promotion Copy

Unlock the full potential of your organoid research with ANT BIO PTE. LTD.’s specialized extraction solutions! Our high-quality kits—including the Cytoplasmic, Nuclear, and Membrane Protein Extraction Kit (abs9346) and Ultra-Pure Total RNA Rapid Extraction Kit (abs60026)—are meticulously optimized to deliver intact, high-yield RNA and proteins from organoids, supporting downstream molecular analyses and functional studies. Paired with our Organoid Passage Buffer (abs9730) and comprehensive organoid medium kits, we provide a complete workflow from culture to extraction, ensuring reproducibility and reliability in every experiment. Whether you’re studying cancer biology, developmental processes, or drug responses, ANT BIO PTE. LTD. is your trusted partner in advancing organoid research. Explore our full range of products today and accelerate your journey from bench to breakthrough!

8. AI Disclaimer

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ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.