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.
- 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.
- 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).
- Assess the quality and quantity by photometric measurements (OD260) and by capillary gel electrophoresis (alternatively agarose gel electrophoresis, see section 3.2.1).