Top 10 Tips For Gene Silencing & Delivery
Gene silencing through the use of siRNA has become a primary tool for characterizing gene involvement in disease states and interactive pathways, there are several methods for preparing and delivering siRNA. AMSBIO can offer a variety of tools and
services including products for generating siRNA, validated
siRNA expression vectors and
oligo sets. Ready-to-use
Lentivirus particles and custom made lentivirus product services and
tailored siRNA specific transfection reagents.
1. Determine the RNAi tools best suited to your application.
With the popularity of siRNA-mediated gene silencing techniques, a wide variety of siRNA kits and reagents have become available.
AMSBIO offer range of solutions from chemically generated siRNA oligos, to
to kits for in vitro generation of siRNAs, to
shRNA viral vectors.
To choose the best reagents for your application, determine whether you need to obtain short-term gene suppression (~3-7 days) or longer-term gene suppression
(e.g., >3-7 days). Also, consider the cells with which you will be working and how difficult they are to transfect, since siRNA oligos and shRNA viral vectors
are easier to deliver into cells than are shRNA plasmids.
2. Perform appropriate positive and negative controls
Some researchers fail to perform appropriate positive and negative controls for RNAi-mediated gene silencing experiments. These should include scrambled
siRNA sequences and transfection reagent alone for negative controls, and validated siRNA's against unrelated genes for positive controls. Studies lacking such
controls will typically not be acceptable for publication.
3. Minimize exposure to RNases.
RNases are everywhere, and they can easily ruin your study. Therefore, extra precautions need to be taken to avoid RNase contamination of precious siRNA
reagents. Typical precautions include pretreatment of all glass and plastic ware with a validated RNase inhibitor, and the utilization of a designated RNase-free work
area. AMSBIO offers NucleoGuard
a universal inhibitor of RNases compatible with downstream applications.
4. Use a transfection reagent optimized for siRNA delivery.
Most available transfection reagents are optimized for plasmid DNA, not siRNA. Therefore, transfection efficiencies for siRNA can be suboptimal, especially
when transfecting primary cells. Use of a siRNA-specific transfection reagent, such as the
GeneSilencer® siRNA Transfection Reagent, is the best way to ensure efficient siRNA delivery in a wide range of cells, with minimal cytotoxicity.
5. Use proper cell culture technique for obtaining optimal transfection efficiency.
Just as with plasmid transfection, siRNA transfection is generally optimal when cells are at their healthiest. This means maintain optimal culture conditions,
minimize stress, avoid contamination, and transfect at mid-log phase (typically 50-70% confluency for adherent dividing cells).
6. Use previously validated siRNA sequences when possible.
Don't reinvent the wheel. Numerous siRNA sequences for a wide variety of genes from many species have been validated and published in the literature.
AMSBIO offer HuSH-29 is pre-designed shRNA with
genome wide coverage of human, mouse and rat.
7. If a validated siRNA sequence for your target gene has not been identified, use siRNA generated against the entire target gene ORF.
Typically, identifying a 22 bp siRNA sequence that provides significant target gene knockdown (e.g., >70%) requires testing 2-4 different chemically
synthesized siRNA oligos, this can be a serious drain on your lab's financial resources. That is why it is a good idea to use a system such as the
Dicer siRNA Generation Kit, to produce a heterogeneous population of siRNAs against your
entire target gene ORF (or a large portion thereof). This method is quicker and less expensive and it allows generation of multiple siRNA pools from one kit.
8. BLAST your siRNA sequence to confirm only one gene is targeted.
It is important to confirm that your siRNA sequence is unique, so that you do not unintentionally knock down any closely related genes. This is easily done
by using NCBI's free BLAST query available at: http://www.ncbi.nlm.nih.gov/BLAST/. Since siRNAs are
exquisitely sensitive, only a single bp mismatch with the target sequence could significantly reduce, if not eliminate, target gene suppression and produce non-specific
9. If you are unsure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM.
The amount of siRNA required to effectively silence a given gene varies significantly with the gene, the siRNA sequence, and the cell type. Therefore,
optimization of siRNA concentration is usually necessary. Again, this assumes that you cannot find experimental details for your gene / cell type already worked
out in the published literature or in manufacturers’ supplied literature.
10. If the initial application of siRNA to your cells does not reduce the level of your target protein sufficiently, consider repeating the
transfection 2-3 times on the same cells over several days.
Some proteins are abundant and long-lived in the cell. Thus, it may be necessary to apply siRNA to your cells two or three times to effectively reduce
the amount of ambient target protein. Simply repeat the transfection 24-48 hours after the initial transfection, and then repeat after an additional 24-48 hours