antisense Protocols

Category: RNAi Technology Protocols

Protocol describes a procedure to anneal sense and antisense siRNA strands to form a duplex.  The duplexes can then be transfected into cultured cells. - [Read Annealing siRNAs to Produce siRNA Duplexes Protocol]

Category: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector.  This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Category: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription. - [Read Basic Information on In Vitro Transcription]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Freezing gealthy cells (> 95% viable) the protocol obtains a culture 80-90% viable 24 hours after thawing and growing on test vial from step 9. Antisense Research Group - [Read Cell Freezing using Liquid Nitrogen N2]

Category: PCR Protocols

Method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type.  The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers.  A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. - [Read Differential Display-PCR Protocol]

Category: Avian Bird Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes.  The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII).  The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

Category: Transfection Protocols

Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids. This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides. The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate. - [Read Transfection of Mammalian Cells with siRNA Duplexes Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids.  This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides.  The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate. - [Read Transfection of Mammalian Cells with siRNA Duplexes Protocol]

Category: Xenopus Protocols: Xenopus Genetics Protocols

Protocols for Xenopus whole mount in situ hybridization. Includes: Protocols for Making Antisense RNA probe (dig labelled); Protocols for In Situ Hyb. - [Read Xenopus Whole Mount In Situ Hybridization Protocols]