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The exquisite properties of Morpholinos contribute to their high success rate, particularly as compared to Phosphorothioates or other unstable or nuclease-sensitive antisense structural types. However, occasionally Morpholino oligos can fail and there are many factors which can contribute to oligo failure, some correctable once determined. Some of the factors which contribute to failure and solutions for many of these problems are listed as responses below to typical researcher concerns.

  • I can't resuspend my oligos
    • Was the oligo fluffy (expanded and dry) when you received it? If not, it may have taken on some moisture, which will make it very difficult to resuspend from this now hardened pellet. To avoid this, if the oligo is to be stored long term we suggest keeping the vial in a desiccator at room temperature. Alternatively, you can aliquot the oligo right away and store the aliquots at room temperature; be sure to heat and vortex before aliquoting. If you are having difficulty resuspending an oligo, autoclave the solution on liquid cycle and remove from the autoclave as soon as it is back to room pressure. Leaving the oligo overnight on a vigorous shaker might also help dissolution. For difficult-to-dissolve oligos, try to make a stock no more concentrated than 0.5 mM (i.e. 600 µL sterile water added to 300 nmol oligo).

    • If the oligo was fluffy and you are still having problems resuspending it, it may be due to high G content or reduced solubility due to an added moiety such as our lissamine fluorescent tag. In these cases, again we suggest that you autoclave the solution on liquid cycle and remove from the autoclave as soon as it is back to room pressure and then vortex. Repeat if necessary. You can determine the concentration of your stock at any time following this protocol.
  • My oligo isn't working at all (no reduction of target protein)
    • Do you have the correct concentration? Your stock oligo should be dissolved in sterile water without DEPC. You can always check the concentration of your oligo in the stock solution by following this protocol.

    1. In the case of microinjection, make sure you are delivering the right concentration. The final concentration inside the embryo should be no less 2 µM. In most cases 2-10 ng injections are necessary to achieve good results in zebrafish.
    2. For Morpholinos delivered with Endo-Porter, start with a Morpholino concentration of 10 µM and try a range of final Endo-Porter concentrations (e;g; 2, 4, 6 and 8 µM) to find the optimal concentration for cytosolic delivery. Check for delivery of fluorescent-labeled oligos by fluorescence microscopy
    3. For Vivo-Morpholinos, make sure your final concentration is greater than or equal to 3 µM for optimal results.

    • Do you have the correct target sequence? Make sure that the sequence of the oligo is the complement of a desirable target sequence. Keep in mind that inaccuracies in RNA sequences in public database files or from 'in-house' sequencing can contribute to improper targeting. Also, make sure if the oligo is a translational blocker that the oligo targets sequence somewhere in the 5' UTR through the first 25 bases of coding sequence. If the oligo is a splice-blocking oligo then the sequence should target a region including a splice junction or a splice-regulatory protein binding site.

    • Are you analyzing antisense activity at the right time? Make sure you are analyzing activity at the appropriate time. For example, if you target an mRNA encoding an enzyme or a transcription factor the degradation of preexisting protein might be rapid enough that you can see a knockdown in 24 hours, whereas if you target an mRNA encoding a ubiquitous structural protein then days might pass before a large enough fraction of the preexisting protein is degraded so that you can detect the knockdown. Morpholino oligos are extremely stable and a knockdown can be assayed a week after delivery as long as it is not diluted by cell division in the organism or cell culture. The decision as to how long to wait before assaying antisense activity is a judgment call made by the researcher. This decision should be made based on the researcher's knowledge of target specifics including: the target mRNA transcript concentration and stability, the protein concentration and stability, the potential for similar or redundant secondary targets, and the possibility of sequence anomalies or errors. Keep in mind that some highly expressed transcripts, like actin, may be difficult to shut down irrespective of oligo concentration.
  • My oligo works, but only reduces expression by 30% or less.
    • In most cases simply delivering more oligo should increase the level of activity. However, keep in mind that specificity is reduced as concentration is increased. For this reason it is important to deliver only enough oligo to achieve near-quantitative shutdown without affecting non-specific targets.

    • There are other factors that can contribute to reduced activity which include potential secondary oligo targets and secondary structure within an oligo.

    1. The secondary targets can come from homologous genes, particularly in the case of tetraploid organisms like Xenopus laevis. However secondary targets can also occur in non-tetraploid organisms when targeting one gene member of a family of homologous genes or even by random sequence similarities.
    2. Secondary structure of an oligo can have a significant impact on activity. However, the concern is much greater for inter-strand pairing between oligos than intra-strand pairing within an oligo since single-molecule stem-loops do not readily form due to the oligo's limited flexibility, but inter-strand pairing can tie-up oligo and thus eliminate it from the pool of oligo that can pair with target sequence. Either way, these concerns are typically not a problem, as oligos designed by Gene Tools should not exhibit significant intra-strand or inter-strand pairing.
  • My oligo used to work great and now it doesn't work anymore.
    • Morpholino oligos are extremely stable. Morpholinos do not degrade in normal storage conditions. Most oligos that have been resuspended in sterile, un-treated water and stored at room temperature do not lose activity. However, some sequences slowly form complexes suspended in water and, over time, do lose antisense activity; activity of complexed oligos can often be restored by autoclaving. Oligos can lose activity if improperly stored or stored in anything other than sterile pure water.

    • It is possible for an oligo to lose activity by a chemical change if they have undergone long-term exposure to acid or have been exposed to diethylpyrocarbonate (DEPC), neither of which are present in oligos as they are delivered to researchers. Gene Tools recommends resuspending oligos in sterile, un-treated water.

    • It is possible for an oligo to fall out of solution if the stock concentration is close to its solubility barrier and it has been chilled or frozen. Gene Tools recommends room temperature storage and heating stocks at 65C for 10 minutes with vortexing or autoclaving prior to aliquotting. The concentration of your stock can be determined at any time by following this protocol.