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 inefficien t 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 Tr ypan 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:


(Date:10/12/2017)... (PRWEB) , ... October 12, 2017 , ... ... Sciences today announced the three Winners and six Finalists of the 2017 Blavatnik ... annually by the Blavatnik Family Foundation and administered by the New York Academy ...
(Date:10/12/2017)... , ... October 12, 2017 , ... ... Wound Market with the addition of its newest module, US Hemostats & Sealants. ... for thrombin hemostats, absorbable hemostats, fibrin sealants, synthetic sealants and biologic sealants used ...
(Date:10/11/2017)... ... 2017 , ... At its national board meeting in North ... the co-founder, CEO and chief research scientist of Minnesota-based Advanced Space Technology and ... ARCS Alumni Hall of Fame . ASTER Labs is a technology development ...
(Date:10/11/2017)... (PRWEB) , ... October 11, 2017 , ... ComplianceOnline’s Medical ... place on 7th and 8th June 2018 in San Francisco, CA. The Summit brings ... well as several distinguished CEOs, board directors and government officials from around the world ...
Breaking Biology Technology:
(Date:5/16/2017)... N.J. , May 16, 2017  Veratad Technologies, ... provider of online age and identity verification solutions, announced ... K(NO)W Identity Conference 2017, May 15 thru May 17, ... Regan Building and International Trade Center. ... the globe and in today,s quickly evolving digital world, ...
(Date:5/6/2017)... 2017 RAM Group , Singaporean ... breakthrough in biometric authentication based on a ... to perform biometric authentication. These new sensors are based ... by Ram Group and its partners. This sensor will ... chains and security. Ram Group is a next ...
(Date:4/19/2017)... , April 19, 2017 The ... vendor landscape is marked by the presence of several ... however held by five major players - 3M Cogent, ... companies accounted for nearly 61% of the global military ... companies in the global military biometrics market boast global ...
Breaking Biology News(10 mins):