RNAi Technology Protocols

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

Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol 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.

Cloning siRNA Duplexes into pSUPER or pSUPERretro Vectors Protocol Protocol for cloning siRNA duplexes into pSUPER or pSUPERretro vectors.

Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples Information on how detect and quantitate MicroRNA in laser capture microdissection samples. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR; Detection of miRNA in Microdissected Tissue from Mouse Brain by qRT-PCR; Differential Expression of MicroRNA in Whole Brain Tissue Compared to a More Homogeneous Population of Cells.

Electroporation and RNAi in the Rodent Retina In Vivo and In Vitro Protocol Protocol for electroporation and RNAi in the rodent retina in vivo and in vitro.

Protocol for siRNA-Primed RNA Synthesis Protocol Short interfering RNAs (siRNAs) can be used to prime RNA synthesis by the RNA-dependent RNA polymerase (RdRP).  SiRNAs can be used by RdRP as primers for specific cellular mRNAs, forming dsRNA products capable of inducing transitive RNAi.

si-RNA Transfection of Mouse Bone Marrow Progenitors by Electroporation Protocol Protocol for si-RNA transfection of mouse bone marrow progenitors by electroporation.

siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection.

siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol II Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection.

The siRNA User Guide The siRNA user guide. Includes: Selection of siRNA duplexes from the target mRNA sequence; Preparation of siRNA duplexes; Useful siRNA reagent combinations; Transfection of siRNA duplexes; Sequences of siRNA duplexes used in our studies.