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AUMantagomir sdASO protocol

In vitro protocol for miRNA inhibition using self-delivering antisense oligonucleotides

AUMantagomir sdASOs enable miRNA inhibition without the need for transfection reagents, in cell lines and hard-to-transfect primary cells.

Protocol overview

AUMantagomir sdASO delivery is a three-step process:
  1. 01Plate cells at optimal density (50-70% confluency at treatment)
  2. 02Add AUMantagomir sdASO directly to culture medium
  3. 03Incubate and analyze results (typically 24-72 hours after treatment)

This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.

Key advantages

  • No transfection required: Add to media (no lipofection, electroporation, or viral vectors needed)
  • Cell compatibility: Works in difficult-to-transfect cell types including primary cells, neurons, and immune cells
  • High specificity: Precisely targets specific miRNAs with minimal off-target effects
  • Low toxicity: Maintains cell viability without transfection reagent-associated toxicity

Materials and reagents

Required items

  • AUMantagomir sdASO (lyophilized or stock solution)
  • Appropriate cell culture medium
  • Culture plates or vessels
  • Mammalian cells of interest
  • Sterile nuclease-free water or buffer (for ASO resuspension)
  • Microcentrifuge tubes (for aliquoting ASO stock)
  • Standard cell culture equipment:
    • Sterile pipettes and tips
    • Cell culture hood
    • Humidified cell culture incubator
    • Centrifuge

Detailed protocol

  1. Step 1: Cell preparation

    Plate cells in their optimum growth medium at a density appropriate for the cell type. A confluency of 50-70% at the time of treatment is typical.

    • For adherent cells: Plate cells the day before treatment, at a seeding density that brings them to that confluency by the time of treatment (or at densities optimized for your specific cell type and assay endpoint). Allow cells to adhere overnight.
    • For suspension cells: Prepare cells at appropriate density shortly before treatment with AUMantagomir sdASO.
    • Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay.

  2. Step 2: AUMantagomir sdASO stock preparation

    Prepare AUMantagomir sdASO stock solution by reconstituting lyophilized ASOs at the desired concentration. If you already have a stock solution prepared, skip to Step 3.

    • Resuspend lyophilized AUMantagomir sdASO using the appropriate volume of sterile nuclease-free water or buffer to achieve the desired stock concentration (typically 1 mM).
    • Pipette the solution up and down 3-5 times while avoiding the introduction of bubbles.
    • Let the vial sit at room temperature for 5-10 minutes to ensure complete resuspension.
    • Centrifuge for 30-45 seconds to collect the solution at the bottom of the tube.
    • Prepare several aliquots of the stock solution to avoid multiple freeze-thaw cycles.

    Important: To avoid degradation, minimize freeze-thaw cycles of your ASO stock. It is strongly recommended to make single-use aliquots of your stock solution and store them at -20°C.

    A clear 24-well plate with pink medium in every well beside an open microtube holding a little solution, a pipette tip entering the tube from above, and three short strands drawn as chains of beads above the tube
    Cells seeded, here in a 24-well plate; the oligonucleotide waits in solution.

  3. Step 3: AUMantagomir sdASO delivery to cells

    Add AUMantagomir sdASO to the cells at the desired final concentration. The recommended working range is 5-20 μM, with a starting concentration of 10 μM. The optimal concentration varies with the target gene, the RNA class (messenger RNA, microRNA or long non-coding RNA) and the cell type, and should be determined by titration for each system.

    • For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMantagomir sdASO, or add the ASO stock directly to the media overlaying the cells. Mix gently.
    • For suspension cells: Either pellet the cells by low-speed centrifugation and gently resuspend the cell pellet in media containing AUMantagomir sdASO, or add the ASO stock directly to the media containing the cells. Mix gently.
    • It is highly recommended to perform a dose response using the three concentrations of the range (5 μM, 10 μM and 20 μM) to determine the optimal concentration for your specific miRNA target.
    • For highly abundant miRNAs or certain cell types, the top of the range, 20 μM, may be required.

    Optimization tip: The optimal ASO concentration may vary depending on the target miRNA abundance, cell type, and assay timing.

    A single-channel micropipette held upright over a 24-well plate of pink medium, its tip releasing one drop into a well
    Add the self-delivering oligonucleotide straight to the medium: no transfection reagent.

  4. Step 4: Incubation and analysis

    Incubate cells with AUMantagomir sdASO and analyze miRNA inhibition at appropriate time points.

    • Return cells to the incubator and maintain under standard culture conditions.
    • Analyze AUMantagomir sdASO-treated cells after the desired time point, typically 24-72 hours post-treatment.
    • miRNA inhibition can be assessed by:
      • Measuring target miRNA levels (qRT-PCR, northern blot)
      • Evaluating de-repression of miRNA target genes (qRT-PCR, Western blot)
      • Monitoring phenotypic changes associated with miRNA inhibition
      • Using luciferase reporter assays containing miRNA binding sites

    Note: Since AUMantagomir sdASO binds to and inhibits miRNAs, a significant reduction in miRNA levels is not always detected by qRT-PCR. The more reliable measure of successful miRNA inhibition is the de-repression of known miRNA target genes, as these should increase in expression when the miRNA is inhibited.

    A benchtop incubator with its outer door and inner glass door open and a 24-well plate of pink medium on the upper of its two wire shelves
    Return the plate to the humidified incubator under standard culture conditions, typically 24-72 hours.

Reference calculations

Making the stock

Reconstitute the vial in nuclease-free water to 1 mM (1 μL per nmol, so 25 μL for a 25 nmol vial). For a 100 μM stock, add 10 μL per nmol.

VialFor 1 mMFor 100 μM
10 nmol10 μL100 μL
25 nmol25 μL250 μL
50 nmol50 μL500 μL
100 nmol100 μL1 mL

Adding the stock to the well

Add the 1 mM stock to the well at 1:200 for 5 μM, 1:100 for 10 μM and 1:50 for 20 μM. At 10 μM that is 1 μL per 100 μL of medium.

PlateMedium in the well5 μM10 μM20 μM
96-well100 μL0.5 μL1 μL2 μL
48-well250 μL1.25 μL2.5 μL5 μL
24-well500 μL2.5 μL5 μL10 μL
12-well1 mL5 μL10 μL20 μL
6-well2 mL10 μL20 μL40 μL

Treatments per vial

At 10 μM, a 25 nmol vial treats 25 wells of a 96-well plate, 5 of a 24-well plate or 2 of a 12-well plate. Counts are rounded down to whole wells.

PlateMedium in the well5 μM10 μM20 μM
96-well100 μL502512
48-well250 μL20105
24-well500 μL1052
12-well1 mL521
6-well2 mL210

Cell density at treatment

Add AUMantagomir sdASO when adherent cells are at 50-70% confluency. Seed suspension cells and primary cells at the density their cell type guide gives.

Tips and troubleshooting

Optimization tips and best practices

Dose optimization

Always perform dose optimization for new cell types or miRNA targets. Start with the three concentrations of the range (5 μM, 10 μM and 20 μM) to determine the optimal balance between inhibition efficiency and economy.

Extended inhibition

For long-term experiments, miRNA inhibition can be maintained for several days using a single dose. For very fast-growing cells, you may need to add more AUMantagomir sdASO to the cell culture every 3 to 5 days to maintain inhibition. Non-dividing cells may not need a further dose for 10 to 14 days.

Appropriate controls

Include a non-targeting control AUMantagomir sdASO in your experiments to distinguish specific effects of miRNA inhibition from any potential non-specific effects of the ASO chemistry.

Target validation

Confirm successful miRNA inhibition by measuring the expression of known miRNA target genes. An effective AUMantagomir sdASO treatment should result in increased expression of genes normally repressed by the target miRNA.

Troubleshooting common issues

Low inhibition efficiency
Increase concentration: Move up towards 20 μM, the top of the range, for highly abundant miRNAs or challenging cell types.
Extend incubation time: Some miRNA inhibition effects may take longer to manifest (48-96 hours after treatment).
Check miRNA expression: Confirm that your target miRNA is expressed in your cell model under your experimental conditions.
Try an alternative readout: If you do not see a reduction in miRNA levels by qRT-PCR, look for de-repression of known target genes or use a reporter assay.
No observable phenotype
Verify inhibition: Confirm that the miRNA is effectively inhibited by measuring target gene de-repression.
Extend observation time: Some phenotypic effects may take time to develop after miRNA inhibition.
Consider redundancy: Other miRNAs from the same family may compensate for the inhibited miRNA. Consider using multiple AUMantagomir sdASOs to target related miRNAs simultaneously.
Check experimental conditions: The phenotypic effect of miRNA inhibition might only be observable under specific conditions (e.g., stress, differentiation, or stimulation).
Cell toxicity
Reduce concentration: If toxicity is observed, move down towards 5 μM, the bottom of the range.
Check cell density: Ensure cells are at optimal density (50-70% confluency) at the time of treatment.
Consider miRNA function: The miRNA might have an essential role in cell survival or proliferation, and its inhibition could naturally cause growth arrest or apoptosis.

Storage and additional information

Storage conditions

  • AUMantagomir sdASOs are shipped in lyophilized form. Upon arrival, store at -20°C.
  • Resuspended AUMantagomir sdASOs should be stored in aliquots at -20°C to avoid multiple freeze-thaw cycles.
  • For short-term storage (up to 1 week), resuspended ASOs can be kept at 4°C.

Additional notes

  • AUMantagomir sdASOs are compatible with standard cell culture media, including those containing serum.
  • No pre-treatment or media change is required before adding AUMantagomir sdASO to cells.
  • AUMantagomir sdASO can be fluorescently labeled to monitor cellular uptake.
  • For phenotypic assays, the timing should be optimized based on both the inhibition kinetics and the turnover of proteins regulated by the target miRNA.

Note

AUMantagomir sdASOs are for research use only. Not for use in diagnostic or therapeutic procedures.

miRNA inhibition assessment

There are several methods to confirm successful miRNA inhibition:

  1. Direct miRNA measurement: qRT-PCR or northern blotting. Note that bound AUMantagomir sdASO may interfere with miRNA detection.
  2. Target de-repression: Measure increased expression of known miRNA target genes by qRT-PCR or Western blot.
  3. Reporter assays: Use luciferase reporters containing miRNA binding sites: effective inhibition will increase reporter signal.
  4. Functional readouts: Assays specific to the biological function of the target miRNA.

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For research use only. Not for use in diagnostic or therapeutic procedures.