Navigation Links
Dicer vs. RNase III for Preparation of siRNA Cocktails


Both recombinant human Dicer and Escherichia coli RNase III can be used in vitro to cleave long dsRNA, producing siRNA cocktails that are effective mediators of gene silencing (1-6). Dicer is an endoribonuclease that contains RNase III domains and is the enzyme responsible for cleavage of long dsRNAs to siRNA in the endogenous RNAi pathway. The siRNAs produced by Dicer are 21-23 bp in length and contain 3' di-nucleotide overhangs with 5'-phosphate and 3'-hydroxyl termini (7-11). E. coli RNase III is involved in the maturation and degradation of diverse cellular, phage, and plasmid RNAs (12-14). Also responsible for digesting long dsRNA, its cleavage products range from ~12 to 15 bp in length with termini identical to those produced by Dicer (3).

Ambion scientists have spent considerable time comparing the silencing effects of Dicer and RNase III generated siRNA cocktails. Although RNase III does produce shorter cleavage products than Dicer, these shorter products are active mediators of gene silencing when transfected into cells (Figure 1; see also Inducing RNAi with siRNA Cocktails Generated by RNase III). Indeed, no difference in efficacy has been found between RNase III and Dicer generated cocktails (Figure 2). Cytotoxic and nonspecific effects are low for both methods (Figures 2B and 3). Because Dicer is more difficult to overexpress and purify than RNase III, its price is usually much higher. In addition, purified Dicer is a rather inefficient enzyme, requiring long digestion times (12-16 hour). In contrast, RNase III digestions take only an hour. For these reasons, Ambion has chosen RNase III for its Silencer siRNA Cocktail Kit (RNase III), which includes reagents for preparing siRNA cocktails to 20 different genes.

Figure 1. 12-15 bp RNase III Digestion Products Elicit Silencing. A 200 bp GAPDH dsRNA (30 g) was digested with RNase III (30 U) for 1 hour at RT. Digestion products were run on a 15% non-denaturing acrylamide gel and the 12-15 bp products were excised, eluted, and ethanol precipitated. A sample was run on a 15% non-denaturing acrylamide gel for visualization (A). HeLa cells were transfected with 100 nM of the 12-15 bp RNase III generated GAPDH siRNAs or a 21 bp chemically synthesized GAPDH siRNA. GAPDH protein levels were monitored by immunofluorescence 48 hours after transfection (B) and the resulting images were quantitated (C).

Figure 2. Potency and Toxicity of siRNA Cocktails.(A) HeLa cells were transfected 24 h after plating with 50 nM, 25 nM, or 12.5 nM final concentration of either RNase III generated, Dicer generated, or chemically synthesized siRNAs to GAPDH. The cells were harvested after 48 h and the reduction in protein levels was examined by immunofluorescence microscopy. Fluorescent signal was quantitated and normalized for cell number. The level of GAPDH in the cells was reduced in a dose dependent manner for all methods of siRNA production, with a 94%, 92%, or 91% reduction being achieved at 50 nM final siRNA concentration, respectively. (B) Duplicate samples from Panel A were examined for cellular toxicity using a Trypan Blue Exclusion Assay. Background levels of cell death of 2% were recorded in nontransfected cells; none of the transfected samples showed significantly higher levels of cellular toxicity.

Figure 3. RNase III Generated siRNA Cocktails Show Specificity for Silencing. HeLa cells were transfected with 100 nM RNase III generated siRNAs to GAPDH. Immunofluorescence analysis of GAPDH, La, c-MYC, Cdk-2, Ku-90, and -actin was performed 48 hours post transfection and subsequently quantitated.


back to top


Ordering Information
Cat# Product Name Size 1625 Silencer siRNA Cocktail Kit (RNase III) 20 rxns
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Protector RNase Inhibitor
2. Protector RNase Inhibitor Enhance the Protection of RNA against Degradation
3. RNase Protection Assay (RPA) Using DIG-Labeled RNA Probes
4. Inducing RNAi with siRNA Cocktails Generated by RNase III
5. Combat RNase Contamination in the Lab
6. Measuring RNase Activity A Real-time Kinetic Analysis
7. RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control
8. Sample Preparation for Microinjection
9. High Pure PCR Template Preparation Kit
10. Proteinase K, Recombinant, PCR Grade: the Ideal Tool for Template Preparations
11. Genomic DNA Preparation from RNAlater Preserved Tissues
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Dicer RNase III for Preparation siRNA Cocktails

(Date:7/30/2014)... San Diego, CA (PRWEB) July 30, 2014 ... second contract from the U.S. Environmental Protection Agency ... company’s expertise in developmental toxicity models, including tests ... on neuron formation and the formation of connections ... function. Inhibition of neurodifferentiation and synapse formation by ...
(Date:7/29/2014)... July 30, 2014 SoundConnect ... collaboration company, is proud to announce the strategic ... positions in their Boston, Charleston and Atlanta offices. ... the increased demand for hosted solutions, collaboration and ... and reduce cost. , SoundConnect was founded in ...
(Date:7/29/2014)... 2014   Sequenom, Inc. (NASDAQ: ... genetic analysis solutions, and Mayo Medical Laboratories (MML), ... the United States , have announced ... applications. "We have great appreciation for Mayo ... we welcome the opportunity to partner with the organization,s ...
(Date:7/29/2014)... has created a new way of manufacturing microstructured ... made by self-assembly of carbon nanotubes, could exhibit ... mechanical stiffness and strength, or the ability to ... have demonstrated that mechanical forces can be used ... and that we can independently control the ...
Breaking Biology Technology:U.S. Environmental Protection Agency (EPA) Awards Vala Sciences a Second ToxCast Contract 2U.S. Environmental Protection Agency (EPA) Awards Vala Sciences a Second ToxCast Contract 3SoundConnect Organization and Culture is Evolving 2Sequenom Enters Into License Agreement With Mayo Medical Laboratories 2Sequenom Enters Into License Agreement With Mayo Medical Laboratories 3Sequenom Enters Into License Agreement With Mayo Medical Laboratories 4A new way to make microstructured surfaces 2
... Key words: somatostatin • ... SPA Imaging Beads , ... with a wide range of biological actions (1–3) and a reduction ... diseases. The SS analog, octreotide (SMS 201–995), is used clinically in ...
... , ... LEADseeker , , ... continually increase screening throughput. Yet accuracy and sensitivity remain vital concerns, ... System was developed to meet these demands by combining excellent detection ...
... into Gems of Knowledge Using discoveryHub™ , ... This paper discusses key problems and opportunities created by ... of life science data available today, and presents a ... rapid ‘harvesting’ and analysis of critical scientific data in ...
Cached Biology Technology:The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System 2The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System 3The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System 4The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System 5Use of the HitHunter EFC Chemiluminescent Detection Kits with LEADseeker Multimodality Imaging System 2Use of the HitHunter EFC Chemiluminescent Detection Kits with LEADseeker Multimodality Imaging System 3Use of the HitHunter EFC Chemiluminescent Detection Kits with LEADseeker Multimodality Imaging System 4Exploiting the Life Science Data Explosion to Speed New Drug Discovery 2Exploiting the Life Science Data Explosion to Speed New Drug Discovery 3Exploiting the Life Science Data Explosion to Speed New Drug Discovery 4Exploiting the Life Science Data Explosion to Speed New Drug Discovery 5Exploiting the Life Science Data Explosion to Speed New Drug Discovery 6Exploiting the Life Science Data Explosion to Speed New Drug Discovery 7Exploiting the Life Science Data Explosion to Speed New Drug Discovery 8Exploiting the Life Science Data Explosion to Speed New Drug Discovery 9Exploiting the Life Science Data Explosion to Speed New Drug Discovery 10Exploiting the Life Science Data Explosion to Speed New Drug Discovery 11Exploiting the Life Science Data Explosion to Speed New Drug Discovery 12Exploiting the Life Science Data Explosion to Speed New Drug Discovery 13Exploiting the Life Science Data Explosion to Speed New Drug Discovery 14Exploiting the Life Science Data Explosion to Speed New Drug Discovery 15Exploiting the Life Science Data Explosion to Speed New Drug Discovery 16Exploiting the Life Science Data Explosion to Speed New Drug Discovery 17
(Date:7/30/2014)... A group of researchers from the University of Helsinki ... experimentally to reproduce in mice morphological changes which have ... gradual modifications in the embryonic development of mice teeth, ... morphologically are very similar to those observed in the ... millions of years ago. , To modify the ...
(Date:7/30/2014)... study of dominance in female baboons suggests that the route ... mom, and to have lots of supportive sisters. , A ... strength, but by the rank of her mother - the ... be. For this reason, dominance rank in female baboons is ... mothers are guaranteed a good spot in the pecking order, ...
(Date:7/30/2014)... from HIV infection could be as simple as inserting ... , University of Washington bioengineers have discovered a potentially ... women from contracting HIV. Their method spins the drug ... with moisture, releasing higher doses of the drug than ... creams. , "This could offer women a potentially more ...
Breaking Biology News(10 mins):Scientists reproduce evolutionary changes by manipulating embryonic development of mice 2Supportive moms and sisters boost female baboon's rank 2Supportive moms and sisters boost female baboon's rank 3Dissolvable fabric loaded with medicine might offer faster protection against HIV 2Dissolvable fabric loaded with medicine might offer faster protection against HIV 3
... involving single gene mutations can reveal a great deal ... not accord with clinical expectations. Australian scientists acknowledge ... new study, which examines people with ,Autosomal Dominant Hyper ... mutation in the STAT3 gene. This makes patients slightly ...
... University of Minnesota have discovered that a human antiviral enzyme ... The discovery, reported in the July 14 issue of ... the enzyme, called APOBEC3B, is responsible for more than half ... Nature in February. APOBEC3B is part of a family ...
... key grassland species from taking hold and hampers efforts ... Work by scientists at Newcastle University, UK, has looked ... how this might inform future restoration work., Although renowned ... petunias or strawberry patch, still relatively little is known ...
Cached Biology News:Surprise finding reveals how adaptive our immune systems can be 2Antiviral enzyme contributes to several forms of cancer, University of Minnesota researchers say 2Grazing slugs hinder grassland restoration 2
Ca2+ Channel Ca gamma 2 Purified Anti-Mouse, Anti-Rat, Anti-Human clone Polyclonal, Isotype Rabbit Ig, 50 µl Consult technical datasheet for details....
Rabbit polyclonal antibody to NCOA2 [Agarose Immobilized]...
Monocarboxylate (lactate) transporter 2...
Mouse Anti-Human Kallikrein 2 (Hk2)...
Biology Products: