What is the recommended purification strategy for siRNA?
In principle, desalting of siRNA is sufficient for an initial siRNA experiment. It is the most economical choice and allows the user to easily identify potent siRNAs. However, to fully exclude effects of -n sequences, HPLC or PAGE purification is recommended.
What is the recommended synthesis scale for siRNA?
Again, an economical choice for trial experiments is to choose our smallest scale: 0.040 µmol. However, once a good siRNA has been identified, 0.2 µmol synthesis scale is favourable, since you will get more siRNA for your money.
Is my siRNA ready to use?
Your siRNA is delivered ready to use, meaning fully deprotected and annealed. If you prefer to obtain the strands separately, please indicate this on the order form.
How should I resuspend my siRNA or single strand RNA?
Use the provided RNase-free water and respend the pellet at a appropriate concentration. Please click here for a more detailed description.
How to design a functional siRNA?
Benefit from Microsynth’s unique advisory service. Give us a call and we help you to design a potent siRNA. As an alternative, use our free online design program.
Which overhang is best for my siRNA?
The most determinant factor for effectiveness is the 19 bp core sequence. Any overhang (RNA or DNA) may be chosen. DNA overhangs may be more nuclease resistant, therefore, Microsynth recommends you to use dTdT overhangs. This is also the most economical choice.
Recent studies suggest that siRNA is very stable once introduced to the cells.
Do you guarantee a siRNA to be effective?
We guarantee that the siRNA looks just perfectly on a Poly-Acrylamide gel. However, we do not guarantee for functionality of your chosen sequence, since this is dependent on multiple factors we can not influence (e. g. protein stability). Guarantees on the mRNA level do not make sense as they neglect the fact that reduction of PROTEIN level is crucial for a phenotype observation.
However, to meet our customer's demands, we now offer a knockdown-guarantee for our siRNAs, if you let us design and synthesize three siRNA for one target gene.
My siRNA does not show an effect. What can I do?
A lot of factors influence the effectivnes of your siRNA. As first step we recommend the following:
Titrate the amount of siRNA used in your experiment. Use a positive control with a simple readout (e.g. GFP).
Optimize transfection/delivery efficiency. It should exceed 90% if you look at a pool of cells.
Change your readout system - e.g. look at the mRNA instead of the protein. Your protein migh have a long half-life.
Re-anneal your siRNA (see how to resuspend siRNAs or click here). Lyophilization might decrease activity.
Make sure that your gene of interest is expressed in your cell line of interest. There may be cell-type specific splice forms.
Check the sequence information in the database in the context of your cell line. Same as above.
Choose a different siRNA. Not all siRNAs are potent down-regulators.
Can my siRNA/RNA be modified? What modifications do you offer?
siRNAs can be modified, preferably (for functional reasons) at the sense strand on the 5’ side.
Most common modifications include FAM, TAMRA, Cy3, Cy5, and Cy5.5 dyes.
Generally, most modifications which we offer for our DNA oligonucleotides are possible for RNA oligonucleotides. Other specialities include 2’-O-Methyl RNA, or terminal 3’-O-Methyl RNA. For more information or for a special request contact the RNA customer service.
Do secondary structures in my RNA influence synthesis?
Yes, they may. There are difficult sequences such as hairpins which may also interfere with deprotection and purification. However, we at Microsynth have vast experience also with difficult sequences.
What is the maximum/minimum length of RNA synthesis?
Maximum length is 65 bases with the 0.2 umol scale (30 bases with the 0.04 umol scale). However, on request we can synthesize longer RNAs. Please contact us for more information. Minimum length is 5 bases. Please contact us for more information.
