10.2.1. siRNA design and synthesis

So far most bee scientists have used dsRNA rather than siRNA for RNAi experiments. Although dsRNA molecules have advantages in handling, off-target effects (Jarosch et al., 2012) have been reported in honey bees. Therefore the usage of siRNAs is recommended where feasible. This allows the selection of one or a few short sequences to initiate RNAi, rather than the many tens of possible permutations generated by a typical dsRNA construct, any of which might cause effects away from the desired target.

  1. Design 3-6 siRNAs for your target gene in order to find an optimal siRNA.
    General guidelines for siRNA design:
    • siRNA targeted sequence is usually 21 nt in length.
    • Avoid regions within 50-100 bp of the start codon and the termination codon.
    • Avoid intron regions.
    • Avoid stretches of 4 or more bases such as AAAA, CCCC.
    • Avoid regions with GC content <30% or > 60%.
    • Avoid repeats and low complex sequence.
    • Avoid single nucleotide polymorphism (SNP) sites.
    • Perform BLAST homology search to avoid off-target effects on other genes or sequences (16- to 18-nt–long stretches of homology are suggested as the maximum acceptable length in RNAi studies per Ambion siRNA design guidelines).
    • Design negative controls by scrambling the target siRNA sequence. This control RNA has the same length and nucleotide composition as the target specific siRNA but in a different order. Make sure that the scrambled siRNA does not show homologies for any known bee gene.
    Several web based programs for appropriate siRNA design, which implement the actual siRNA design algorithms, are available (e.g. siRNA target designer version 1.6 (Promega); siDesign center (Dharmacon, Inc); Block-iTTM RNAi Designer (Invitrogen).
  2. Use T7 RibomaxTM Express RNAi System (Promega) for siRNA production.
    1. Follow the manufacturers´ instructions.
    2. Incubation time may be increased in order to increase the siRNA yield (A time-course experiment has to be performed beforehand in order to find the optimal incubation time).
  3. Assess the quality and quantity by photometric measurements (OD260) and by capillary gel electrophoresis (alternatively agarose gel electrophoresis, see section 3.2.1).