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MISSION® siRNA FAQ's

N-TER Transfection

Can I use N-TER to transfect in my plasmid DNA?
No, N-TER will not work with plasmid DNA.
 
Can I use N-TER to transfect siRNA into HUVEC cells?
Yes, please see the protocol we offer on the website at http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/huvec_protocol.pdf.
 
Can I use the N-TER peptide alone as a negative control?
No, use of the N-TER peptide alone may result in cellular cytoxicity. We recommend that you use a buffer-only control and a non-targeting siRNA for negative controls. We offer non-targeting siRNA controls as Catalog numbers SIC001 and SIC002. These controls have been validated for use in human, mouse, and rat cells.
 
Can you compare N-TER to lipid-based transfection reagents?
The use of N-TER™ results in slightly higher assessed knockdown: In a head-to-head comparison, 25 gene targets were examined: 24 gene targets expressed higher knockdown when N-TER was used vs. a competitor's lipid-based transfection reagent as the transfection reagent. Viability was comparable where 7 of 25 targets demonstrated higher viability when a competitor's lipid-based transfection reagent was used. The mean overall difference in viability was only 7.1%. Average increase in knockdown was 22% using N-TER vs. a competitor's lipid-based transfection reagent at 5 ul. When a competitor's lipid-based transfection reagent was used at a higher concentration, viability was impacted: 19 of 25 targets showed decreased viability by an average of 13% when compared to N-TER-treated cells. However, even when increased amounts of a competitor's lipid-based transfection reagent were use, knockdown continued to be greater using N-TER in 18 of 25 instances.
 
N-TER is a peptide-based transfection system. Lipid-based reagents are typically targeted to endosomal compartments which can result in the production of inflammatory cytokines and global changes in gene expression (an off-targeting effect). Additionally, targeting of siRNA to the endosome can result in siRNA degradation. N-TER bypasses endosomal compartments resulting in less off-targeting effects and less degradation of the siRNA product.
 
Does N-TER target the siRNA to the nucleus or the cytoplasm?
The N-TER peptide has been modified so that it releases the siRNA to the cytoplasm. Cytoplasmic release of siRNA has been shown to correlate with increased knockdown efficiency.
 
Does the N-TER bind the siRNA through a covalent reaction?
No, N-TER associates with the siRNA in a non-covalent manner. While covalent attachment can increase the uptake of siRNA species, release of the covalently attached siRNA can be a barrier for efficient and rapid knockdown. Non-covalently attached siRNA is released very rapidly into the cytoplasm of transfected cells resulting in rapid mRNA knockdown.
 
How do I scale-up my transfection reaction?
N-TER/siRNA complex formation is less efficient in large volumes. We recommend that you scale up in 3X volumes. These 3X volumes can be combined after complex formation has taken place.
 
Do you have any references for N-TER?
Yes, the updated list is at sigma.com/nter.