How to Process Epithelial Organoids and Organoid-Derived Epithelial Monolayers for RNA Isolation

The following protocols describe the method for processing epithelial organoids (section I) and organoid-derived epithelial monolayers (section II) from different tissue types, including intestinal, lung, pancreatic, liver, kidney, and stomach, for RNA isolation.


Materials

  • RNeasy Mini Kit (Qiagen Catalog #74106)
  • β-Mercaptoethanol (e.g. Sigma Catalog #516732)
  • TRIzol™ Reagent (Thermo Fisher Catalog #15596026)
  • DMEM/F-12 with 15 mM HEPES (Catalog #36254)
  • D-PBS (Without Ca++ and Mg++) (Catalog #37350)
  • Advanced DMEM/F-12 (Thermo Fisher Catalog #12634-010)
  • Falcon® Conical Tubes, 15 mL (Catalog #38009)
  • Nuclease-Free Water (Catalog #79001)
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    • Linear Acrylamide (Invitrogen™ Catalog #AM9520)
    • Glycogen, RNA grade (Thermo Scientific™ Catalog #R0551)
    • Chloroform (Sigma Catalog #C2432)
    • Isopropanol (Sigma Catalog #I9516)
    • Optional: QIAshredder homogenizers (Qiagen Catalog #79656)
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Considerations before starting

  1. Indicated volumes are for organoids cultured in 30 - 50 μL Matrigel® (Corning 356231) domes cultured in 24-well plates.
  2. For organoids cultured in suspension, transfer the entire organoid and medium volume from a culture well to a 15 mL conical tube.
    • Centrifuge tubes at 250 - 300 x g for 5 minutes at room temperature (15 - 25°C). Carefully aspirate supernatant.
    • Proceed to step 3 of the protocol.
  3. Remove the culture plate containing the organoids from the incubator and check dome integrity and organoid health under the microscope prior to processing.
  4. 3 - 5 healthy, confluent culture wells per condition will typically yield ~50 - 500 ng/μL of RNA, depending on the tissue type.
    • Low RNA yield can be expected from a single organoid dome, which typically contains 150 - 200 organoids. When preparing for RNA extraction, we suggest seeding higher-density domes and seeding approximately three domes at that density. It is not recommended to extract RNA from a single dome.
  5. Optional: Supplement an aliquot of Buffer RLT (from RNeasy Mini Kit) with 10 μL of 14.3 M β-mercaptoethanol (β-ME) per mL of Buffer RLT. RLT + β-ME solution can be stored at room temperature (15 - 25°C) for up to 1 month.

Note: Cells can be harvested for applications like qPCR by simply removing the medium and lysing the cells in RNA lysis buffer, e.g Buffer RLT from Qiagen or TRIzol™, then immediately storing at -80°C or processing to purify the RNA. This is the fastest method that exposes the cells and RNA to minimal stress and degradation.

Protocol

I. RNA Isolation from Epithelial Organoids Using RNeasy Mini Kit:

  1. Check that the domes to be harvested are intact. If domes are loose, proceed to step 3.
  2. Aspirate spent medium from wells.
  3. Forcefully add 1 mL of ice-cold DMEM/F-12 + 1% BSA or cold Advanced DMEM/F-12 + 1% BSA directly on top of the domes.
    Note: For intestinal organoids, 1 mL of cold PBS can be used. This cold wash will help remove excess Matrigel®.
    Note: Adding BSA will reduce the loss of material from organoids adhering to plasticware.
  4. Triturate organoids with a 1 mL pipettor to break up the Matrigel® domes and fragment the organoids.
    Note: Trituration can be adjusted or modified depending on the organoid type. As a general guideline, triturate organoids 3 - 5 times and check under a microscope. Organoids should be ideally broken down into fragments.
    Note: Specific guidelines for trituration
    • Intestinal organoids: 20 times
    • Pancreatic organoids: 2 - 3 times
    • Hepatic organoids: 15 times
  5. Transfer replicates of each condition into one 15 mL conical tube. Rinse wells with 0.5 mL of organoid dissociation solution (the same solution used in step 3) and transfer to respective 15 mL conical tube.
  6. Centrifuge at 300 x g for 5 minutes at room temperature (15 - 25°C).
  7. Aspirate the supernatant, taking care to not disturb the pellet.
    Notes:
    • After centrifugation, residual Matrigel® may be observed as a layer on top of the pellet of organoid fragments. This may occur when the organoid dissociation solution is not cold enough. Be careful when aspirating the layer of Matrigel® to avoid disturbing the cell pellet.
    • If there is a translucent pellet above the organoid pellet, this is evidence that the Matrigel® might not be fully depolymerized. If so, aspirate the supernatant, add a fresh cold solution (e.g DMEM / F-12), and break up the pellet using a P1000 pipette. Add an additional 1 mL of cold solution and centrifuge at 300 x g for 5 min at 4°C.
  8. Once the Matrigel® has been eliminated, aspirate the supernatant. Resuspend each pooled condition in 350 µL of Buffer RLT + β-ME to lyse the organoid pellet as per the Qiagen kit protocol. Triturate the cell lysate until all visible organoid fragments are dissolved and the mixture first acquires a viscous consistency, then becomes slightly less viscous.
    Note: RNA clean-up can be carried out using the RNeasy Mini Kit.
    Note: Protein lysis buffer can be added to the pellet for subsequent western blot analysis.
    Note: For Matrigel®- or extracellular matrix (ECM)-free protocols (e.g. PneumaCult™ Apical-Out Airway Organoid Medium), start from step 5. Centrifuge organoids to collect and lyse (see step 6).
  9. Optional: If organoid fragments remain, centrifuge lysate through a QIAshredder homogenizer to fully break up tissue. Transfer the entire lysate volume to a QIAshredder spin column placed in a 2 mL collection tube. Centrifuge the spin column at 10,000 x g for 2 minutes at room temperature. The spin column can be discarded. Cap the collection tube containing the homogenized lysate.
  10. Transfer lysate into labeled collection tubes on ice.
    Note: The lysate can be stored at -80°C for up to 6 months prior to RNA isolation. Immediately store the sample at -80°C.
  11. Proceed to RNA isolation as per the manufacturer’s instructions.
  12. Elute RNA in 30 μL of nuclease-free water.
  13. Quantify final RNA concentration with a nano-drop or bioanalyzer and store at -80°C.

II. Alternative Protocol for RNA Isolation from Epithelial Organoids Using TRIzol™:

The TRIzol™ protocol could be considered if attempting RNA isolation from a smaller organoid sample (for example, less than 3 - 5 culture wells).

Note: Please refer to your institution’s guidelines for the safe disposal of phenol, chloroform, or TRIzol™.

  1. Remove medium from organoids, replace with cell recovery solution (Corning® Cell Recovery Solution, at least 350 μL/well), and place on ice for at least 15 minutes.
    Note: Multiple wells can be pooled, if necessary. Depending on the confluency of domes, 2 - 3 Matrigel® domes are suggested.
  2. Resuspend and pool each well in a 2 mL Eppendorf tube (or larger centrifuge tube to accommodate larger volumes). Centrifuge at 300 x g for 5 minutes at 4°C.
  3. Remove as much supernatant as possible and replace with chilled PBS.
  4. Repeat steps 2 and 3 at least 2 additional times.
  5. Resuspend organoids with 1 mL TRIzol™ reagent. Pipette up and down several times to mix and homogenize cells.
    Note: TRIzol™ can be added directly to the Matrigel® domes after removing spent medium from organoid wells (steps 1 - 4 in this protocol are optional).
    Note: If pooling, 1 mL TRIzol™ can be used for processing multiple domes of the same condition. The rest of the protocol can be adjusted per 1 mL of TRIzol™.
  6. Incubate at room temperature (15 - 25°C) for 5 minutes.
  7. Add 0.2 mL of chloroform per 1 mL of TRIzol™.
  8. Secure the lid tightly and shake vigorously for 10 - 15 seconds.
  9. Incubate for 2 - 3 minutes at room temperature.
  10. Centrifuge at 12,000 x g for 15 - 30 minutes at 4°C.
    Note: In some cases, clear separation does not occur after 15 minutes, and longer centrifugation is needed.
  11. Gently collect the clear, aqueous upper layer and transfer to a fresh RNAse-free 1.5 mL Eppendorf tube. Be careful not to disturb the red phenol-chloroform lower layer or white precipitate in the interphase.
  12. Add 1 μL of 20 mg/mL RNAse-free glycogen. Alternatively, add 2 μL of a neural carrier (Linear Acrylamide 5 mg/ml) per sample.
  13. Add 0.5 mL of isopropanol. Incubate at 4°C for 10 minutes.
  14. Centrifuge at 12,000 x g for 15 minutes, at 4°C.
  15. Discard the supernatant.
  16. Wash the pellet in 1 mL cold 75% ethanol and centrifuge at 12,000 x g for 5 minutes at 4°C. Repeat this step.
  17. Discard the supernatant with a pipette and air dry for 5 -10 minutes. Do not dry entirely.
  18. Resuspend in 50 μL of RNAse-free dH2O.
  19. Treat with RNAse-free DNase I following manufacturer protocols.
  20. Repeat steps 5 - 20, if necessary.
  21. Quantify the final RNA concentration with a nano-drop or bioanalyzer for 230/280 and 260/230 ratios and store at -80°C.

III. RNA Isolation from Organoid-Derived Epithelial Monolayers Using RNeasy Mini Kit:

RNA extraction from organoid-derived epithelial monolayers can be challenging. The following steps can be followed for RNA isolation from different organoid-derived epithelial monolayers grown as submerged monolayers or as air-liquid interface (ALI) cultures:

  1. Remove medium from the wells.
  2. If using transwell inserts, apply 350 μL/insert of Buffer RLT + β-ME from the RNeasy Mini Kit directly to the membrane to lyse cells. If using standard tissue culture-treated plates, add the lysis reagent directly to the culture dish.
    Note: Volume of lysis buffer can be adjusted based on the cultureware and cell density.
    Multiple wells can be pooled, if necessary.
  3. Transfer lysate into labeled collection tubes on ice (e.g. 15 mL conical tubes)
    Note: The lysate can be stored at -80 °C for up to 6 months prior to RNA isolation. Immediately store the sample at -80°C if not proceeding with RNA isolation.
  4. Proceed to RNA isolation as per the manufacturer’s instructions.
  5. Elute RNA in 30 μL of nuclease-free water.
  6. Quantify the final RNA concentration with a nano-drop or bioanalyzer and store at -80°C.

Tips for Improving Yield with Organoid-Derived Epithelial Monolayers

  • Use TRIzol™ rather than the RNeasy Mini Kit for detaching cells from the membrane (at risk of stressing the cells; please refer to the TRIzol™ Reagent User Guide for dissociating cells grown in monolayers).
  • Concentrate the sample by reducing the volume of lysis buffer or increasing the amount of culture used (number of wells/number of organoids)
  • Document #PR00066
  • Version 1.0.0
  • November 2022