Before preparing siRNA, it is necessary to design the siRNA sequence separately. Studies have found that for mammalian cells, the most effective siRNAs are double-stranded RNAs with a size of 21-23 bases and two protruding bases at the 3' end; while for non-mammalian cells, long fragment dsRNAs are more effective. The sequence specificity of siRNA is very strict, and a single base mismatch with the target mRNA will significantly weaken the effect of gene silencing.
Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA with 20 to 25 nucleotides and has many different uses in biology. siRNA has a short double-stranded RNA (dsRNA) with a phosphorylated 5' end and a hydroxylated 3' end with two prominent nucleotides. This structure is processed using an enzyme called dicer, which can cut longer double-stranded RNA or small hairpin RNA into siRNA. siRNAs are known to be involved mainly in the RNA interference (RNAi) phenomenon, regulating gene expression in a specific manner. In addition, they are also involved in a number of RNAi-related response pathways, such as antiviral mechanisms or changes in chromatin structure. However, the response pathways of these complex mechanisms are not yet understood.
1. The design of siRNA is a critical first step in determining the success or failure of RNAi experiments.
Not every siRNA is effective in triggering gene silencing. As with primer design, there are rules for designing effective siRNAs that have been learned from previous experience and are being refined as siRNA research progresses.
2. The 3' end of the antisense strand of siRNA preferably ends in UU, which is recognized as the most efficient siRNA structure.
siRNAs ending in other bases may also successfully trigger RNAi. However, if the siRNA is synthesized by in vitro transcription, avoid ending with G, because RNase will cut off the G at the end during subsequent processing.
3. Multiple siRNA sites are preferably dispersed over the full length of the mRNA.
There is no data to show that the position of siRNA on the mRNA has any special relationship with the effect of RNAi. If siRNA sites affect siRNA effects because of mRNA secondary structure or protein-binding shielding, spreading out the distribution can reduce the risk.
4. Avoid homologous sequences with other genes.
All potential target sequences were subjected to BLAST at NCBI, and sequences with base homology to other genes must be avoided.
5. 30%-50% GC ratio has a higher success rate than siRNAs with high GC content.
When there are many choices, be careful not to have more than 3 consecutive GCs, and try to avoid a series of certain bases.
6. Design at least 3-5 or more siRNAs for a target gene and conduct parallel experiments to improve the success rate.
It has been assessed that randomly designed siRNAs have a 25% chance of effectively silencing gene expression (reducing more than 75%-95% of mRNA) and more than half the chance of achieving 50% silencing.
Starting from the transcriptional AUG start codon, the downstream AA sequence was searched for and the 19 nucleotides adjacent to the 3' end of each AA were recorded as candidate siRNA target sites.
Potential sequences are compared with the corresponding genomic database (human, or mouse, rat, etc.) to exclude sequences that are homologous to other coding sequences/ESTs.
A complete siRNA experiment should have a negative control. The siRNA used as a negative control should have the same composition as the selected siRNA sequence, but no obvious homology to the mRNA. It is common practice to disrupt the base sequence in the selected siRNA. Of course, it is also important to ensure that it is not homologous to other genes.
Customer provided
The accession number of the target gene (or gene sequence)
The success of RNAi experiments relies heavily on siRNA design, as sequence specificity determines effective gene silencing. A single base mismatch can significantly reduce knockdown efficiency.
We start from the AUG start codon and identify candidate 19-nucleotide sequences with optimal features for silencing. Multiple candidate sites are evaluated to maximize efficiency.
Effective siRNAs usually end with UU at the 3' antisense end, maintain 30-50% GC content, and avoid homology with other genes. Dispersing multiple siRNA sites across the mRNA also improves silencing success.
Designing 3-5 or more siRNAs per target allows parallel testing and increases the likelihood of identifying the most effective sequence. This approach improves overall experiment success rates.
Negative controls are sequences with similar composition but no homology to the target mRNA. This ensures observed effects are specific to the designed siRNA.
