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Protocol

AUMantagomir toASO protocol

In vitro protocol for miRNA inhibition using transfection-optimized antisense oligonucleotides

AUMantagomir toASO are transfection-optimized antisense oligonucleotides for microRNA inhibition in mammalian cell culture. They are the same designs at a lower price, delivered with a transfection reagent, and are used for high-throughput screening and routine microRNA inhibition in standard cell lines.

Protocol overview

AUMantagomir toASO delivery requires standard transfection methods. This protocol covers several approaches:
  1. 01Lipid-based transfection (most common method)
  2. 02Electroporation (for difficult-to-transfect cells)
  3. 03Calcium phosphate transfection (economical option)
  4. 04Polymer-based transfection

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

Key advantages

  • Cost-effective: A lower price than the self-delivering products, for high-throughput studies and large-scale experiments
  • High specificity: Minimal off-target effects compared to siRNA approaches
  • Flexible delivery: Compatible with all standard transfection methods
  • Same AUMsilence platform: Maintains the specificity and the stability of AUMantagomir sdASO

Materials and reagents

Required items

  • AUMantagomir toASO (lyophilized or stock solution)
  • Appropriate cell culture medium
  • Serum-free medium (for lipid-based transfection)
  • Culture plates or vessels
  • Mammalian cells of interest
  • Sterile nuclease-free water or buffer (for ASO resuspension)
  • Microcentrifuge tubes (for aliquoting ASO stock)
  • Transfection reagent of choice:
    • Lipid-based transfection reagent
    • Electroporation system (if using electroporation)
    • Calcium phosphate transfection reagents (if using this method)
    • Polymer-based transfection reagent
  • 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 transfection is typical.

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

  2. Step 2: AUMantagomir toASO stock preparation

    Prepare AUMantagomir toASO 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 toASO using the appropriate volume of sterile nuclease-free water or buffer to achieve the desired stock concentration (typically 100 μM).
    • 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.
    • Prepare two working stocks from that one by diluting aliquots in sterile nuclease-free water, because a reconstituted stock is too concentrated to pipette accurately at the volumes a well needs at 50 nM to 100 nM. A 2 μM stock, a 1 in 50 dilution of it, for the lipid-based route, where the oligonucleotide is diluted into serum-free medium. And a 25 μM stock, a 1 in 4 dilution, for the calcium phosphate and polymer routes, where it goes into a small fixed volume whose proportions the chemistry depends on.

    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.

  3. Step 3: Transfection method selection

    AUMantagomir toASO can be delivered using various transfection methods. Choose the most appropriate method for your cell type and experimental needs.

    • Lipid-based transfection: Most common and versatile method, suitable for a wide range of cell types.
    • Electroporation: Effective for difficult-to-transfect cells, including primary cells and some immune cells.
    • Calcium phosphate transfection: Economical option suitable for some adherent cell lines.
    • Polymer-based transfection: Alternative method that works well for certain cell types.

    Selection tip: If you are unsure which method to use, lipid-based transfection is recommended as a starting point for most cell types. For difficult-to-transfect cells, electroporation often provides the best results.

  4. Step 4: Lipid-based transfection protocol

    This protocol describes a general lipid-based transfection method for AUMantagomir toASO. Always refer to the specific instructions provided by the manufacturer of your transfection reagent.

    • Dilute AUMantagomir toASO: In a sterile tube, dilute AUMantagomir toASO in serum-free medium to achieve the desired concentration. For a 24-well plate, use 50 μL of serum-free medium and 15-30 μL of the 2 μM working stock, which gives a final concentration in the well of 50 nM to 100 nM, the recommended range.
    • Dilute transfection reagent: In a separate sterile tube, dilute the transfection reagent in serum-free medium according to the manufacturer's recommendations. For a 24-well plate, typically use 50 μL of serum-free medium and 1-3 μL of transfection reagent.
    • Combine and incubate: Add the diluted transfection reagent to the diluted AUMantagomir toASO (not the reverse). Mix gently by pipetting up and down or tapping the tube. Incubate at room temperature for 10-20 minutes to allow complexes to form.
    • Add complexes to cells: Add the transfection complex solution dropwise to the cells. Gently rock the plate to ensure even distribution.
    • Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing inhibition efficiency.

    Note: Some transfection reagents require a media change 4-6 hours post-transfection to minimize toxicity. Refer to the manufacturer's guidelines for your specific transfection reagent.

  5. Step 5: Electroporation protocol

    Electroporation is an effective method for delivering AUMantagomir toASO to difficult-to-transfect cells. The exact parameters will depend on your electroporation system and cell type.

    • Prepare cells: Harvest cells in the growth phase and wash with PBS. Resuspend cells in the appropriate electroporation buffer at a density recommended for your electroporation system (typically 1-5 × 106 cells/mL).
    • Add AUMantagomir toASO: Add AUMantagomir toASO to the cell suspension to achieve a concentration of 1-2 μM in the electroporation buffer. Mix gently.
    • Electroporate: Transfer the cell/ASO mixture to electroporation cuvettes. Perform electroporation using parameters optimized for your cell type (commonly used settings: 200-450V, 1-3 ms pulse length).
    • Recovery: Immediately after electroporation, add pre-warmed complete medium to the cells. Transfer the cells to culture plates or flasks.
    • Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing inhibition efficiency.

    Important: Electroporation parameters vary significantly between cell types and electroporation systems. Always optimize the electroporation conditions for your specific cells and equipment.

  6. Step 6: Calcium phosphate transfection protocol

    Calcium phosphate transfection is an economical method suitable for some adherent cell lines.

    • Prepare Solution A: In a sterile tube, mix AUMantagomir toASO (1.1-2.2 μL of the 25 μM working stock for a 24-well plate) with 125 mM CaCl2 solution to a total volume of 25 μL.
    • Prepare Solution B: In a separate tube, add 25 μL of 2X HBS buffer (HEPES-buffered saline, pH 7.05-7.12). It is the same volume as Solution A, which is what brings the 2X HBS to 1X where the precipitate forms; move one of the two and the other moves with it.
    • Form precipitate: Add Solution A dropwise to Solution B while gently vortexing or bubbling air through Solution B using a pipette. Incubate at room temperature for 20-30 minutes to allow precipitate to form.
    • Add to cells: Add the transfection mixture dropwise to cells in complete medium. Gently rock the plate to ensure even distribution.
    • Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing inhibition efficiency.

    Note: The pH of the 2X HBS is critical for efficient transfection. Small variations in pH can significantly affect transfection efficiency. So is the 1X the equal volumes give it, and so is the calcium: at the volumes above the oligonucleotide is <10% of Solution A and the mix carries about 57 to 60 mM calcium.

  7. Step 7: Polymer-based transfection protocol

    Polymer-based transfection is an alternative method that works well for certain cell types.

    • Dilute AUMantagomir toASO: In a sterile tube, dilute AUMantagomir toASO in the buffer recommended by the polymer transfection reagent manufacturer. For a 24-well plate, use 50 μL of buffer and 1.1-2.2 μL of the 25 μM working stock, which keeps the oligonucleotide a small part of that buffer rather than a third of it.
    • Dilute polymer reagent: In a separate sterile tube, dilute the polymer transfection reagent in the appropriate buffer according to the manufacturer's recommendations.
    • Combine and incubate: Add the diluted polymer reagent to the diluted AUMantagomir toASO. Mix gently and incubate at room temperature for the time specified by the manufacturer (typically 10-15 minutes).
    • Add complexes to cells: Add the transfection complex solution dropwise to the cells. Gently rock the plate to ensure even distribution.
    • Incubate cells: Return the cells to the incubator and maintain under standard culture conditions for 24-72 hours before assessing inhibition efficiency.

    Important note: Always follow the specific instructions provided by the manufacturer of your polymer-based transfection reagent, as protocols can vary significantly between products.

  8. Step 8: Incubation and analysis

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

    • Return cells to the incubator and maintain under standard culture conditions.
    • Analyze AUMantagomir toASO-treated cells after the desired time point, typically 24-72 hours post-transfection.
    • 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 toASO 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.

Reference calculations

Making the stock

Reconstitute the vial in nuclease-free water to 100 μM (10 μL per nmol, so 50 μL for a 5 nmol vial). Prepare the 2 μM and 25 μM working stocks from it (Step 2).

VialFor 100 μM
2 nmol20 μL
5 nmol50 μL
10 nmol100 μL
25 nmol250 μL

Volumes for lipid-based transfection

Cell culture plate96-well24-well12-well6-well
AUMantagomir toASO (2 μM working stock)13-6 μL15-30 μL30-60 μL63-125 μL
Serum-free medium for ASO dilution10 μL50 μL100 μL250 μL
Transfection reagent20.2-0.6 μL1-3 μL2-6 μL5-15 μL
Serum-free medium for reagent dilution10 μL50 μL100 μL250 μL
Complete medium in well100 μL500 μL1 mL2 mL

Table notes

  1. These volumes give 50 nM to 100 nM in the well (the complete medium plus the two dilutions). The working stock and the reagent add a little volume, which lowers the final concentration by a few per cent.
  2. The amount of transfection reagent can vary significantly depending on the specific product and cell type. Always refer to the manufacturer's recommendations.

Transfections per vial

At 100 nM, a 5 nmol vial transfects 416 wells of a 96-well plate, 83 of a 24-well plate or 41 of a 12-well plate. The volume in the well is the complete medium plus the two dilutions. Counts are rounded down to whole wells.

PlateVolume in the well50 nM100 nM
96-well120 μL833416
24-well600 μL16683
12-well1,200 μL8341
6-well2,500 μL4020

Cell density at transfection

Transfect adherent cells with AUMantagomir toASO when they 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

Optimize transfection conditions

For each new cell type or transfection reagent, optimize the ASO:reagent ratio and total concentration. Test 2-3 different ratios to find the optimal balance between inhibition efficiency and cell viability.

Cell density matters

Cell confluency at the time of transfection significantly impacts efficiency. For lipid-based transfection, 50-70% confluency is typically optimal. Too low or too high cell density can reduce transfection efficiency.

Serum considerations

Some transfection reagents are inhibited by serum. If recommended by the manufacturer, perform the transfection in serum-free medium and replace with complete medium 4-6 hours post-transfection.

Antibiotics

For sensitive cells, consider removing antibiotics from the medium during transfection, as the combination of transfection reagents and antibiotics can increase toxicity.

Appropriate controls

Include appropriate controls in your experiments: untreated cells, cells treated with transfection reagent only, and a non-targeting control AUMantagomir toASO 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 toASO treatment should result in increased expression of genes normally repressed by the target miRNA.

Troubleshooting common issues

Low transfection efficiency
Optimize ASO:transfection reagent ratio: Test different ratios to find the optimal balance.
Check cell density: Ensure cells are at the appropriate confluency (typically 50-70%) at the time of transfection.
Verify transfection reagent quality: Some reagents lose potency over time or with multiple freeze-thaw cycles.
Try a different transfection method: If lipid-based transfection yields poor results, consider electroporation or polymer-based transfection.
Move within the recommended range: The recommended final concentration in the well is 50-100 nM. If the first attempt was at 50 nM, raise it to the top of that range before changing anything else. If inhibition is still poor there, the thing to reconsider is the design rather than the dose: try a second sequence against the target.
High cell toxicity
Reduce transfection reagent amount: Excessive transfection reagent can be toxic to cells.
Change medium post-transfection: Replace the transfection medium with fresh complete medium 4-6 hours after transfection.
Remove antibiotics: For sensitive cells, antibiotics in the medium can increase transfection-related toxicity.
Reduce ASO concentration: If toxicity persists, try decreasing the ASO concentration while maintaining the optimal ASO:reagent ratio.
Consider target biology: If your target is essential for cell survival, apparent toxicity may be a consequence of successful inhibition.
Low inhibition efficiency
Extend incubation time: Some miRNA inhibition effects may take longer to manifest (48-96 hours after transfection).
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 toASOs 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).

Storage and additional information

Storage conditions

  • AUMantagomir toASO are shipped in lyophilized form. Upon arrival, store at -20°C.
  • Resuspended AUMantagomir toASO 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 toASO are optimized for standard transfection methods but do not self-deliver like AUMantagomir sdASO.
  • Always follow the specific instructions provided by the manufacturer of your transfection reagent, as protocols can vary significantly between products.
  • 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 toASO 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 toASO 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.