MISSION ® siRNA

Experimental Design FAQs

 
 
Do you have an easy way to do reverse transfections of siRNA with N-TER?
Yes, please see the protocol we offer on the website at http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Brochure/nter_sirna_nanoparticles.pdf.
 
Can siRNA integrate into the genome so that I can get stable knock down cell lines?
No, siRNA cannot integrate into the genome. If you need stable "knocked down" cell lines, please see the shRNA product line.
 
How long does siRNA silence a gene?
Generally, gene silencing resulting from siRNA can be assessed as early as 24 hours post transfection. The effect most often will last from 5-7 days. However, duration and level of knockdown are dependent on cell type and concentration of siRNA. Transfections may be repeated to maintain knockdown.
 
How can I enhance the effect of my siRNA?
It has been demonstrated* that adding enoxacin to siRNA experiments caused an increase in the ability of the siRNA to knock down the target gene. For more information, see this paper.
*Shan, G., et al., A small molecule enhances RNA interference and promotes microRNA processing. Nature Biotechnology 26: 933-40 (2008).
 
Another reference which may be helpful is: Duxburya, M.S., et al., RNA interference: A mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells. Biochemical and Biophysical Research Communications 331(2): 459-463 (2005). 
 
Do you offer pooled siRNAs?
Yes, in two different formats.

siRNA pools contain 5nmol per duplex and can be comprised of both predesigned and custom siRNA duplexes. For more information: sigma.com/missionsirna

MISSION esiRNA, or Endoribonuclease-prepared siRNAs, are pools of siRNA oligos resulting from cleavage of long double-stranded RNA (dsRNA) with Escherichia coli RNase III. Since MISSION esiRNAs are pools of siRNAs that all target the same mRNA sequence, they are highly specific. esiRNA are available in 0.1nmol, 0.25nmol, 2nmol or 5nmol quantities.

For more information: sigma.com/esirna 
 
Do you have a recommended procedure for pooling individually ordered duplexes?
Yes. For each duplex that will be pooled, remove half of the quantity from each siRNA duplex, following the steps below.

  1. Resuspend each duplex with 100uL of molecular biology grade water (Product # W4502) (no need for buffer, it is already included in the dry material)
  2. Remove 50uL from each of the component siRNA and transfer to one new tube.
  3. The remaining siRNA solution in each tube can be used for further evaluation or additional studies. Recommended storage temperature is -20 °C.
 
At what concentration should I make my siRNA stock solution?
For the stock solution, we recommend a concentration of 50-100 µM.
 
Can I resuspend the dried siRNA duplex pellet in RNase-free water instead of 1x TE?
Actually, we do not recomend resuspending dried siRNAs in 1x TE at all.
 
Before reconstituting dried siRNAs, briefly spin the tubes. siRNA is dried down in the presence of buffer (listed below), therefore, the siRNAs should be resuspended in molecular biology grade water (DNase- and RNase-free), Sigma-Aldrich catalog number W4502.
 
siRNA Buffer:
Potassium Acetate (100 mM)
HEPES (30 mM)
Magnesium Acetate (2 mM)
 
Note: siRNAs are susceptible to degradation by nucleases introduced during handling. Use RNase-free reagents, tubes, and filtered/barrier pipette tips. Always wear gloves when handling siRNA products.
 
How do I calculate the concentration of the siRNA in my sample?
RNA is most accurately quantified by measuring its absorbance at 260nm (A260) with a dual beam spectrophotometer.
Use Beer’s Law: A260 = (e)(C)(L)
e= the extinction coefficient (provided on the QA document)
C = the siRNA concentration
L = the path length of the cuvette
The final concentration of the resuspended siRNA may be calculated by solving for C and multiplying by the dilution factor.
 
How do I convert nmol to ug?
Multiply the number of moles by the MW on the Quality assurance document.
 
As an example, if you received 10 nmol of siRNA, and the MW of that siRNA is 13,300, calculate as follows:
(10 nmol)(13,300 g/mol)(mol/10^9 nmol)(10^6 µg/g) = 133 µg
 
How do ODs relate to nmols and ug?
siRNA is a dsRNA:
1 A260 unit = 60 ug dsRNA
1 OD siRNA duplex = 5 nmol = 60 ug
 
How much siRNA do I use for various plate/well sizes? How many wells of a 6 well plate can I transfect with 2 OD (10 nmol) siRNA?
Please see table for suggested reagent amounts. NOTE: Final siRNA concentration = 30nM    1 nmol = 1000 pmol
 
Typical siRNA experiment reagent amounts
Format Transfection Reagent (µL) siRNA (pmol) cells / well Final Volume (mL)
96 well 0.3-1 3 6000 0.1
24 well 1-3 15 40000 0.5
12 well 2-4 30 80000 1.0
6 well 3-6 75 200000 2.5
In answer to your second question, you can transfect about 10000/75 = ~130 wells
 
What siRNA concentration(s) should be used when starting a new experiment?
We recommend testing siRNAs in small pilot experiments to validate the best concentration for every cell type and new experimental procedure, using a concentration range from 5 nM to 100 nM MISSION siRNAs in culture medium. We find that 30 nM is typically a reasonable starting siRNA concentration. Ideally, the lowest concentration that results in the desired knockdown should be used. This will also help reduce potential off-target effects.
 
If I am overexpressing a gene in my cells, will siRNA to that gene knock it down?
In numerous instances overexpressed genes have been shown to be successfully knocked down using siRNA.
 
While the siRNA should chew away at some of the message being generated; it's hard to say what the exact effects will be. You may need to balance final siRNA concentration with any toxicity/cellular innate immunity response to see functional knockdown.
 
The gene I want to knock down is very stable. How will this affect my siRNA experiment?
Even stable genes, such as the common housekeeping genes like GAPDH and Cyclophilin B are effectively silenced using typical siRNA approaches. While performing cell viability analysis is always a good control, this becomes more important when pursuing the silencing of potentially essential genes that may be represented by those most stably expressed.
 
MISSION siRNA has been shown to be quite effecitve at knocking down high-expression, stable messages. While the protein of the gene may be quite stable, the stability of the mRNA should not be a huge factor when using siRNAs. The RNAi process directly targets mRNA transcripts for cleavage, and subsequent degradation by cellular nucleases. Always attempt to optimize the correct amount of siRNA to your message of interest for proper empirical determination.
 
What is the best strategy to elucidate individual siRNA sequences from a heterogeneous population (pooled siRNA)?
If you purchased a pool of synthetic siRNA, rather than pooling the individual siRNAs yourself, the best method would be to order the siRNAs contained in the pool as individuals and test the individuals in your assay. We sell our siRNAs as individuals in concentrations appropriate for pooling and testing individuals.
 
How do I transfect suspension cells?
Sigma-Aldrich does not offer a transfection reagent appropriate for suspension cells. There are several transfection reagents on the market that will work for suspension cells.
 
Do you have a protocol and/or recommended reagents for siRNA transfection?

There is not one single protocol for siRNA transfection that we would be able to post here. It really depends on the transfection reagent you choose to use. We do offer two siRNA transfection reagents.  The MISSION® siRNA transfection reagent, catalog number S1452, for transient knockdown of eukaryotic gene expression and   N-TER™ a Nanoparticle siRNA Transfection System, Sigma-Aldrich catalog number N2913.  N-TER works only for adherent cells, and is not appropriate for suspension cell lines. However, in some cases, if the cell line can be stressed or differentiated and the cells start to adhere, then N-TER may work. This has been observed with U937 cells. These cells cannot be transfected with N-TER in suspension, but if they are stressed and start to stick, they can then be transfected with N-TER.

Both transfection reagents have a posted Technical Bulletin on the product ordering page describing the procedure for transfecting siRNA.

If you are using another transfection reagent, please refer to the supplier’s technical bulletin for the protocol

How do I optimize transfection conditions for my cell line?
To identify optimal transfection conditions for a particular cell line, we recommend setting up a matrix of cell density and siRNA concentration. It is a good idea to use a validated and highly effective siRNA for optimization. A matrix of serum versus no serum should also be tested if appropriate for the cell line. Regardless of the cell type, the N-TER/siRNA complexes will enter the cell quickly, so there is no advantage to incubating the cells longer. Most adherent cell lines can be transfected with N-TER regardless of whether they are immortalized or primary.
 
What cells should I use for siRNA experiments?
Cell lines are often selected as a function of the type of investigation to be conducted. For example, investigations focusing on liver function will often use HepG2 or comparable cell lines since these lines are derived from liver cells.
 
Which cell lines have been tested with your siRNA?
Most of our studies have been conducted in the HeLa and A549 cell lines as these two cell lines are commonly used throughout the community.
 
What should I use for negative control?
We offer MISSION siRNA Universal Negative Controls, and the product numbers are SIC001 and SIC002 for regular siRNA. These controls are available in package sizes of either 1 nmol or 5 x 1 nmol. We can also synthesize negative controls as requested by the customer.  Many customers have their own scrambled sequence that they prefer to use, and some customers use luciferase or eGFP siRNA controls. These can be ordered the same as our custom siRNA, under "Custom Products".
 
The advantage with the luciferase (or other marker genes) is you can use these siRNAs also as positive controls when co-transfected with the respective marker gene.
 
What siRNA controls do you recommend?
For positive controls, we offer several for human and rat. Please visit our positive control webpage at http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/sirna/mission-positive.html. The positive controls are available in our standard siRNA sizes of 10 nmol, 25 nmol, or 50 nmol.
 
We also offer MISSION siRNA Universal Negative Controls, and the product numbers are SIC001 and SIC002 for regular siRNA. These controls are available in package sizes of either 1 nmol or 5 x 1 nmol.
 
We can also synthesize positive or negative controls as requested by the customer as a custom order. Many customers have their own scrambled sequence that they prefer to use, and some customers use luciferase or eGFP siRNA controls. These can be ordered the same as our custom siRNA, under "Custom Products".
 
The advantage with the luciferase (or other marker genes) is you can use these siRNAs also as positive controls when co-transfected with the respective marker gene.