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View additional product information for Custom RNA, Stealth - FAQs (10620312)
12 product FAQs found
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
RNAi oligos can generally be stored lyophilized at -20°C and remain stable for at least 6 months.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
We currently offer the following standard modifications for RNA oligos:
- 5' phosphate
- 5' biotin
- 5' fluorescein
Note: 5' phosphate and 5' biotin are only available as desalted.
Additional modifications may be available by special order if the quantities in your order are large enough - please inquire.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
For single-stranded RNA oligos, the Certificate of Analysis (COA) contains information on the mass, moles, and OD numbers for each individual oligo.
For RNA duplexes, the amount of product delivered is according to the specifications mentioned during ordering the oligos, and is listed on the tube. The COA will still provide details on each single RNA strand.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Synthesis of the RNA oligo is monitored through trityl analysis. Final testing is done by mass spectroscopy to further ensure the quality.
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The coupling efficiency for RNA oligos is 98.5%
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Check the RNAi product pages for the most updated information, but historically the sequence length limit for Thermo Fisher Scientific synthetic RNA oligonucleotides has been 50 bases.
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HPLC purification means that the oligo is purified for full-length (end result will by >95% full length).
Desalting means that chemical by-products of synthesis are removed from the oligo, but no purification of failure sequences is involved.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
The default purity of RNA oligos is Desalted, using a resin that will only remove salts. Alternatively, the RNA can be purified by analytical HPLC which will remove truncated failure sequences and give higher chemical purity as well. We do not offer additional purification post-annealing of duplexes.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
The chemistry of RNA synthesis is identical to DNA synthesis except for the presence of an additional protecting group at the 2' hydroxyl position of ribose. This position is protected with silyl groups, which are stable throughout the synthesis. The remaining positions on both the sugar and the bases are protected in the same fashion as in DNA.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.