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esiRNA Technical Overview
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MISSION esiRNA provides RNAi researchers with a proven, cost-effective, and simple way to perform RNAi screens. MISSION esiRNAs are endoribonuclease-prepared siRNA pools comprised of a heterogeneous mixture of siRNAs that all target the same mRNA sequence. These multiple silencing triggers lead to highly specific and effective gene silencing with lower off-target effects than single or pooled siRNAs.
Individual esiRNA — esiRNAs provided in tubes at either 20 ug or 50 ug quantities targeting human or mouse genes
esiFLEX esiRNA — esiRNAs provided in 96-well customized plates at either 1 ug, 2.5 µg or 5.0 ug targeting human or mouse genes
Plated esiRNA — esiRNAs provided in 96-well customized plates at either 20 ug or 50 ug quantities targeting human or mouse genes
Genome Scale esiRNA Libraries — Two Genome-scale collections of individual esiRNAs including 16,744 human or 14,068 mouse esiRNAs at either 1 ug, 2.5 ug or 5.0 µg quantities in 384-well plates
Long Non-Coding RNA esiRNA Libraries — Two esiRNA libraries targeting non-coding transcripts including 643 esiRNAs from the mouse genome and 1,245 esiRNAs from the human genome..
esiOPEN esiRNA — Custom esiRNAs targeting the species specific 500 base pair gene sequence provided, supplied in tubes at either 20 μg or 50 μg quantities
esiSEC esiRNA — Secondary independent esiRNA for any human and mouse primary esiRNA, supplied in tubes at either 20 μg or 50 μg quantities
How are MISSION esiRNAs prepared?
esiRNA or Endoribonuclease-prepared siRNAs are pools of siRNAs resulting from cleavage of long double-stranded RNA (dsRNA) with Escherichia coli RNase III. Please see Figure 1.
Figure 1: Overview of MISSION esiRNA Production.
Note: This is for the production of an individual esiRNA
(click image to enlarge).
Generation of PCR products
The template for in vitro transcription is generated by PCR amplification of the cDNA from the clone using primers specific for the vector backbone or target-specific primers appended with RNA-Polymerase promoter sequences. The PCR product is sequence verified. The amplified region is selected based on the highest possible number of highly effective siRNA based on the DEQOR siRNA design program. MISSION esiRNA are designed to cover all known transcript variants of the target gene.
In vitro transcription and the production of long dsRNA
The PCR product generated from the cDNA is used for in vitro transcription reactions. The dsRNA is produced utilizing RNA Polymerases and the two single RNA strands are subsequently annealed before digestion.
The long dsRNA is enzymatically digested to short dsRNAs. This digestion produces complex pools of siRNA-like molecules.
Purification of digested dsRNA
The digestion is then purified to remove any remaining DNA template, unincorporated nucleotides, and dsRNAs longer than approximately 40 bp. This purification step results in MISSION esiRNAs with an average length of 21 bp.
Since the starting material is cDNA, each MISSION esiRNA is guaranteed to target a real gene, no more wasted time on assays for "predicted" genes that do not actually exist.
MISSION esiRNA are a heterogeneous mixture of siRNAs that all target the same mRNA sequence. These multiple silencing triggers lead to highly specific and effective gene silencing.
esiRNA FAQ (Frequently Asked Questions)
MISSION esiRNA and MISSION esiFLEX are available individually, as custom arrayed panels, or genome scale libraries that target both human and mouse genes.
Our individual esiRNAs are ideal for the biological characterization of single genes. Currently, we offer esiRNAs targeting 16.744 human and 14.068 mouse transcripts at quantities of either 20 µg or 50 µg normalized to 200 ng/µl; larger scales are available upon inquiry. All esiRNAs are designed based on sequence annotations from ENSEMBL database and packaged in single tubes.
esiRNAs provided in 96-well customized plates at quantities of either 20 ug or 50 ug normalized to 200 ng/µl. Currently, we offer esiRNAs targeting 16.744 human and 14,068 mouse transcripts. Plate orders must have ≥10 targets.
Genome Scale esiRNA Libraries
Our genome scale esiRNA libraries are state-of-the-art RNAi reagents especially suitable for large-scale loss-of-function screening. Genome-scale collections of individual esiRNAs for human or mouse species are available. Currently, we offer a human library composed of 16.744 esiRNA and a mouse library with 14.068 esiRNAs. All esiRNAs are designed based on sequence annotations from the ENSEMBL database. The libraries are delivered in 384-well plates with all positions occupied at either 1 µg, 2.5 µg or 5.0 µg quantities normalized to 50 ng/µl. Other quantities, custom arrays or other plate formats are available upon inquiry.
Our esiFLEX sub-genomic collections of esiRNAs provide a convenient small scale option with plate position customization. We provide esiFLEX for human or mouse species and offer 16,744 human and 14,068 mouse esiRNAs in our portfolio. The esiFLEX libraries are provided in 96-well plates at either 1 µg, 2.5 µg or 2.5 µg quantities normalized to 50 ng/µl. The position of each esiRNA on the plates can freely be chosen. Other quantities, concentrations or plate formats are available upon inquiry. All esiRNAs are designed based on sequence annotations from the ENSEMBL database. Minimum order of 24 targets required.
esiOPEN provides access to fully flexible custom-targeting esiRNAs. Provide any transcript-sequence (minimum 500 bp) regardless of the species and an esiRNA will be synthesized against the best target-region (determined by DEQOR) that lies within the provided sequence to ensure maximum efficiency and specificity. esiOPEN esiRNAs are offered at either 20 µg or 50 µg scales normalized to 200 ng/µl and packaged in single tubes. Larger scales are available upon inquiry.
esiSEC provides secondary independent silencing triggers for any primary esiRNA in our portfolio. esiSEC are used for the validation of RNAi-phenotypes induced by our primary esiRNAs. esiSEC esiRNAs are offered at either 20 µg or 50 µg scales normalized to 200 ng/µl and packaged in single tubes. Larger scales are available upon inquiry. All esiRNAs are designed based on sequence annotations from the ENSEMBL database.
esiRNA Ordering Information
For all other esiRNA product offerings:
MISSION esiRNA — Proven RNAi Screening Tool
Roguev, A. et al. Quantitative genetic-interaction mapping in mammalian cells. Nature Methods 10, 432–437 (2013).
Abbasi, M., Lavasanifar, A. & Uludag, H. Recent attempts at RNAi-mediated P-glycoprotein downregulation for reversal of multidrug resistance in cancer. Medicinal research reviews 33, 33-53 (2013).
Alvarez-Calderon, F., Gregory, MA. & DeGregori, J. Using functional genomics to overcome therapeutic resistance in hematological malignancies. Immunologic research 55, 100-15 (2013).
Kim, N. & Song, K. KIFC1 is essential for bipolar spindle formation and genomic stability in the primary human fibroblast IMR-90 cell. Cell structure and function (2013).
Ngondo-Mbongo, RP., Myslinski, E., Aster, JC. & Carbon, P. Modulation of gene expression via overlapping binding sites exerted by ZNF143, Notch1 and THAP11. Nucleic acids research (2013).
Vella, P. et al. Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells. Molecular cell 49, 645-56 (2013).
Calegari, F. et al. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. PNAS, 99, 14236-14240 (2002).
Lin, Z. et al. (2011) Reduced Level of Ribonucleotide Reductase R2 Subunits Increases Dependence on Homologous Recombination Repair of Cisplatin-Induced DNA Damage. Mol Pharmacol doi:10.1124/mol.111.074708. Abstract
Krastev, D.B. et al. A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Nat Cell Biol 13, 809-18 (2011). Abstract
Nitzsche, A. et al. RAD21 cooperates with pluripotency transcription factors in the maintenance of embryonic stem cell identity. PLoS One 6, e19470 (2011). Abstract
Leushacke, M. et al. An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression. PLoS One (2011), accepted for publication.
Slabicki M., et al. (2010) A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia. PLoS Biol 8(6): e1000408. doi:10.1371/journal.pbio.1000408. Paper (2.22 Mb PDF)
Collinet, et al. Systems survey of endocytosis by multiparametric image analysis. Nature 464, 243-249 (2010). Abstract
Theis, M. et al. Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division. EMBO J. 28, 1453-65 (2009). Abstract
Ding, L. et al. A Genome-Scale RNAi Screen for Oct4 Modulators Defines a Role of the Paf1 Complex for Embryonic Stem Cell Identity. Cell Stem Cell. 9, 403-15 (2009). Abstract
Kittler, R. et al. Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat. Cell Biol. 9, 1401-12 (2007). Abstract
Kittler, R. et al. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036-40 (2004). Abstract
Additional esiRNA Literature References
Stewart, G. S. et al. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136, 420-34 (2009). Abstract
Lawo, S. et al. HAUS, the 8-Subunit Human Augmin Complex, Regulates Centrosome and Spindle Integrity. Curr Biol., 19, 816-26 (2009). Abstract
Fazzio, T. G. et al. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 2008 134, 162-74 (2008). Abstract
Konstantinova, I. et al. EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets. Cell 129, 359-70 (2007). Abstract
Nikolova, G. et al. The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation. Dev Cell. 10, 397-405 (2006). Abstract
Kittler, R. et. al. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. Proc. Natl. Acad. Sci. U.S.A. 102, 2396-401 (2005). Abstract
Liu, W. Y. et al. Efficient RNA interference in zebrafish embryos using synthesized with SP6 RNA polymerase. Dev. Growth Differ. 47(5), 323-31 (2005). Abstract
Yang, D. et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 99, 9942-7 (2002). Abstract
For technical questions concerning esiRNAs, please email our technical Service department at firstname.lastname@example.org
MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.