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Gene Silencing by dsRNA in vivo : RNA Interference
Gene interference is an effect similar to loss-of function mutations in organisms, as if the gene being studied were inactivated. Both sense and antisense RNA are already known to produce interference with the expression of the genes they correspond to by blocking protein synthesis.
Another mechanism for gene interference using RNA has been developed in the past few years. This process, called RNA interference (RNAi) involves double-stranded RNA (dsRNA), and was first developed for use in invertebrates, later vertebrates, and now after much doubt, has been proved to work for mammals, specifically mice.
RNAi proves to be a very good alternative to the gene "knockout" method, in which homologous recombination is used to produce an organism with a disrupted version of a gene.
The RNAi method was developed in 1998, using Caenorhabditis elegans, a type of nematode" (Fire, et. al., 1998).
siRNA a new tool in RNA interference
Duplexes of 21-nucleotide (nt) RNAs with symmetric 2-nt 3' overhangs are introduced into a cell acting as guides to direct the RNA degradation machinery to the target RNAs.
Tuschl and al. have systematically analyzed the silencing efficiency of siRNA duplexes as a function of the length of the siRNAs, the length of the 3’-overhangs, and the sequence in the overhangs.
Using this technique, Elbashir et al, have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines - specifically in HeLa cells.
Xiaomei Bai and al. have recently identified the cDNA for GAG GalTII and report that it is the previously described_1,3-N-acetylglucosaminyltransferase by using two small interfering RNAs (siRNA GalTII-A and -B) obtained from GENSET OLIGOS.
References
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 (1998)
T. Tuschl, P. D. Zamore, R. Lehmann, D. P. Bartel, P. A. Sharp, "Targeted mRNA degradation by double-stranded RNA in vitro." Genes Dev., 13, p3191-3197 (1999).
S. M. Elbashir, W. Lendeckel, T. Tuschl, "RNA interference is mediated by 21- and 22-nucleotide RNAs." Genes Dev., 15, p188-200 (2001).
S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells." Nature, 411, p494-498 (2001).
Xiaomei Bai, Dapeng Zhou, Jillian R. Brown, Brett E. Crawford, Thierry Hennet and Jeffrey D. Esko, "Biosynthesis of the Linkage Region of Glycosaminoglycans." The journal of biological chemistry Vol. 276, No. 51, Issue of December 21, pp. 48189–48195 (2001).
We offer Ready-to-use siRNA – deprotected - Single Strand and Duplex forms
PROLIGO is proud to be the worldwide supplier of licensed siRNA
• siRNA oligos are delivered deprotected and desalted
• in Single Strand or Duplex form.
• Duplex form guaranteed by sending you a photo of the double stranded siRNA on a gel.
• From 1 OD up to 50 OD.
• Purification by HPLC or PAGE available on request.
• TAT : 6 to 9 working days
• Systematically QC controlled
SPECIFICATIONS
| siRNA package |
Quantity OD |
Purification |
Format |
2’-deprotected |
Aliquoting |
TAT days |
| Single strand |
2; 5; 10;50 |
crude |
Dry |
X |
Free on request |
6 |
Duplex
(guaranteed)
|
2; 5; 10;50 |
crude |
Liquid |
X |
Free on request |
7 |
| Single strand |
1; 5; 10; 50 |
PAGE/HPLC |
Dry |
X |
Free on request |
8 |
Duplex
(guaranteed)
|
1; 5; 10; 50 |
PAGE/HPLC |
Liquid |
X |
Free on request |
9 |
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Modifications commonly used for siRNA:
• 5’ –end or 3’ –end labelled siRNA
• 5’ labels : fluorescein, biotin, amine, phosphate, 6-Fam, Hex, Tet.
• 3’ labels : fluorescein, biotin, amine, phosphate.
SPECIAL DUPLEX PRICE & labelled siRNA : contact us at oligos@generalbiosystem.com
Annealing protocol
Annealing Buffer: 10mM Tris, pH 7.5 - 8.0, 50mM NaCl, 1mM EDTA
Mix complementary oligos (at equimolar concentration) in a 1.5ml microfuge tube and add appropriate volume of annealing buffer according to the final concentration of annealed sample you need to reach.
Place tube in a standard heatblock at 90 - 95ºC. Remove the heatblock from the apparatus and allow to cool to room temperature (or at east below 30ºC) on the workbench. Slow cooling to room temperature should take 45-60 minutes. Store on ice or at 4ºC until ready to use.
An alternative procedure for annealing involves the use of a thermal cycler.
Dispense 100m l aliquots of the mixed oligos into PCR tubes (500m l size).
Do not overlay the samples with oil. Place the tubes in a thermal cycler and set up a program to perform the following profile:
(i) heat to 95ºC and remain at 95ºC for 2 minutes, (ii) ramp cool to 25ºC over a period of 45 minutes, (iii) proceed to a storage temperature of 4ºC.
Briefly spin the tubes in a microfuge to draw all moisture from the lid.
Pool samples into a larger tube, store on ice or at 4ºC until ready to use.
Long Term Storage
It may be necessary to aliquot and lyophilize the annealed sample. After drying, the sample may be stored at -20ºC in a desiccated container. Resuspend the annealed oligos at the desired concentration with sterile distilled water. The annealed pair of oligonucleotides is ready for use.
Design your siRNA
Please look at Tuschl and co-workers recommendations : http://www.mpibpc.gwdg.de/abteilungen/100/105/
siRNA are licenced by the Massachusetts Institute of Technology to US Patent Applications 60/265232, 09/821832 and PCT/US01/10188, RNA Sequence-Specific Mediators of RNA Interference.
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