Invitrogen™ Rat Set

The easiest way to do this is to use the identification numbers (HSS, RSS, MSS) and go to “Stealth Quick Order” link online (https://rnaidesigner.thermofisher.com/rnaiexpress/quickOrder.do?icid=fr-rnaiordering-5). Sequences that were provided after purchase can also be re-ordered as “custom” stealth molecules online.

Yes, although you must use specific transfection methods and reagents to optimize the reaction. The procedure below is recommended to cotransfect your plasmid DNA and an RNAi molecule into mammalian cells using Lipofectamine 2000. A more detailed version can be found on our website by searching "RNAi Transfection Protocols".

1. One day before transfection, plate cells in the appropriate amount of growth medium without antibiotics such that they will be 80-90% confluent at the time of transfection.
2. For each transfection sample, prepare DNA-RNAi molecule-Lipofectamine 2000 complexes as follows:
a. Dilute the DNA and RNAi molecule in the appropriate amount of Opti-MEM I Medium without serum. Mix gently.
b. Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in Opti-MEM I Medium without serum. Mix gently and incubate for 5 minutes at room temperature.
c. After the 5 minute incubation, combine the diluted DNA and RNAi molecule with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur. The solution may appear cloudy, but this will not impede the transfection.
3. Add the DNA-RNAi molecule-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
4. Incubate the cells at 37°C in a CO2 incubator until you are ready to harvest cells and assay for your target gene. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.

To find out more about Stealth RNAi, please go to http://www.thermofisher.com and search for "Stealth RNAi". Stealth RNAi molecules are 25 base-pair double-stranded RNA oligonucleotides with proprietary chemical modifications developed to overcome some common limitations of traditional siRNA. Stealth RNAi often eliminates nonspecific effects such as the PKR/interferon stress response caused by siRNA. Nonspecific effects such as stress responses result in growth inhibition and cytotoxicity, making RNAi results extremely difficult to interpret. Stealth RNAi is the only modified dsRNA that effectively avoids recognition by complexes that initiate cellular stress responses, while offering highly potent gene knockdown via RNAi pathway. Our online RNAi Designer, available at https://rnaidesigner.invitrogen.com/rnaiexpress/, applies the most up-to-date rules for Stealth RNAi design, increasing your chances of obtaining high levels of gene inhibition. It is an easy way to design and order any custom synthetic molecule for RNAi.

We recommend resuspending the single-stranded RNA oligo in 1X TE buffer (10 mM TrisCl, pH 8.0, 0.1 mM EDTA), prepared under RNAse-free conditions. This buffers the pH and chelates metal ions that could contribute to RNA degradation. The RNA oligo can also be resuspended in RNAse-free water instead of TE buffer.

Duplex RNA (siRNA) comes lyophilized in 10 mM Tris-HCL, pH 8.0, 20 mM NaCl, 1 mM EDTA which can then be resuspended in the appropriate amount of DEPC-treated water to bring the RNA concentration to 20 uM.

RNAi oligos can generally be stored lyophilized at -20°C and remain stable for at least 6 months.