siRNA Design Services

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.

Introduction to siRNA

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.

siRNA Oligonucleotide Design Principles

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.

Our Services

1) Selection of siRNA target sites

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.

2) Sequence homology analysis

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.

3) Design of negative controls

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)

Features and Advantages

• Covers all human, mouse, and rat gene targets
• Guaranteed knockdown efficiency
• Multiple modifications for higher stability and transfection efficiency