By Muhammad Sohail
content material: Gene silencing by way of RNA interference and the position of small interfering RNAs --
fundamentals of siRNA layout and chemical synthesis --
Oligonucleotide scanning arrays within the layout of small interfering RNAs --
siRNA construction by way of in vitro transcription --
construction of siRNAs with the applying of deoxyribozymes --
creation of siRNA in vitro via enzymatic digestion of double-stranded RNA --
Plasmid-mediated intracellular expression of siRNAs --
Lentiviral vector-mediated supply of si/shRNA --
Exogenous siRNA supply: protocols for optimizing supply to cells --
RNAi in drosophila mobile cultures --
RNAi in caenorhabditis elegans --
supply of RNAi reagents in C. elegans by means of microinjection --
RNAi in drosophila embryos --
RNAi in xenopus laevis oocytes --
RNAi in xenopus embryos --
An RNAi-based genomic library for ahead genetics within the African trypanosome --
RNAi within the malaria parasite plasmodium --
Silencing the expression of cysteine proteases (falcipains) in plasmodium falciparum utilizing RNA interference methods --
RNAi in transgenic animal versions --
RNA interference in mouse types --
Virus-induced RNA silencing in vegetation --
RNA silencing in crops: biolistic supply of RNAi reagents --
Agro-infiltration: a flexible device for RNAi stories in plants.
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Content material: Gene silencing by way of RNA interference and the position of small interfering RNAs -- fundamentals of siRNA layout and chemical synthesis -- Oligonucleotide scanning arrays within the layout of small interfering RNAs -- siRNA construction by means of in vitro transcription -- creation of siRNAs with the appliance of deoxyribozymes -- construction of siRNA in vitro by means of enzymatic digestion of double-stranded RNA -- Plasmid-mediated intracellular expression of siRNAs -- Lentiviral vector-mediated supply of si/shRNA -- Exogenous siRNA supply: protocols for optimizing supply to cells -- RNAi in drosophila mobilephone cultures -- RNAi in caenorhabditis elegans -- supply of RNAi reagents in C.
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Additional info for Gene silencing by RNA interference : technology and application
2 mM MgCl2) (Dharmacon, Inc) ii. 6, 1 mM EDTA) iii. 0 iv. pmd 29 7/26/2004, 1:26 PM 30 Gene Silencing by RNA Interference Experimental Procedures 1. Resuspend the 2'-ACE protected oligomers in 2'-deprotection buffer. If purchased as a duplex, re-suspend the duplex in 400 µl of 2'-deprotection buffer. If purchased as single strands, combine equimolar concentrations of the complementary oligomers into a single microcentrifuge tube. 2. Vortex and centrifuge. 3. Incubate at 60°C for 45 min to cleave the 2'-ACE protecting groups, converting the RNA into the free 2'-OH form (the acid-catalyzed hydrolytic byproducts of the 2'deprotection step are ethylene glycol and formic acid that are volatile and readily removed upon drying and/or desalting).
Arrow: full-length (uncleaved) IGF1R1-1581 transcript. Asterisk: region selected for array screen (modified from Bohula et al. J. Biol. , 278, 15991, 2003; with permission). 4 outlines the method of array fabrication. The synthesis is carried out using standard nucleotide-CEphosphoramidites on an adapted ABI DNA synthesizer (Applied Biosystems). Reagents are delivered to the substrate surface using a diamond-shaped or a circular mask. The mask is sealed against the substrate to create a cell into which reagents for first base synthesis are delivered.
1 While these same studies determined that the penultimate base in the overhang may contribute to target identification, more recent work suggests that the dinucleotide motif and the choice of 3' UU or TT overhangs have no functional consequence. In general, AA(N19) or NA(N19) target motifs can also be found in the 5' or 3' UTRs of the transcript. 11 However, because the UTRs tend to be more polymorphic or to contain conserved regulatory sequences, target sites in these regions should be selected carefully.