Making a Morpholino Experiment Work: Controls, Favoring Specificity, Improving Efficacy, Storage, and Dose

Methods Mol Biol. 2017:1565:17-29. doi: 10.1007/978-1-4939-6817-6_2.


A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.

Keywords: Aggregation; BLAST; Coinjection; Compensation; Concentration; Controls; Dose; Efficacy; Humidor; Hypochromic effect; Lyophilization; MALDI-TOF; Microinjection; Mispair; Morpholino; Nonsense-Mediated Decay; Phenocopy; Rescue; Specificity; Storage; Synergy; Tm; Turnover; Vehicle-only; Vivo-Morpholino; p53.

Publication types

  • Review

MeSH terms

  • Animals
  • Base Pairing
  • Gene Knockdown Techniques
  • Gene Targeting
  • Humans
  • Microinjections
  • Morpholinos / administration & dosage
  • Morpholinos / chemistry
  • Morpholinos / genetics*
  • Morpholinos / standards
  • Oligonucleotides, Antisense / administration & dosage
  • Oligonucleotides, Antisense / chemistry
  • Oligonucleotides, Antisense / genetics*
  • Oligonucleotides, Antisense / standards
  • Preservation, Biological / methods
  • Quality Control
  • RNA Stability


  • Morpholinos
  • Oligonucleotides, Antisense