Encyclopedia of Experiments: Biological Techniques
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Transcript
For reverse transfection of small interfering RNA, or siRNA, a class of double-stranded RNA, into non-adherent adipocytes or fat cells, prepare a suspension of siRNAs targeting a specific protein-encoding gene in adipocytes. Add a suitable cationic lipid-based transfection reagent. Incubate the mixture to form transfection complexes through electrostatic interactions.
Transfer the mixture to collagen-coated wells of a multi-well plate. The transfection complexes immobilize on the plate coating by non-specific interactions. Pipette a suspension of adipocytes into wells containing immobilized transfection complexes. The adipocytes loosely adhere to the plate coating, increasing the physical contact between the cells and complexes.
The transfection complex fuses to the cell membrane and delivers the siRNA into the cytoplasm. The siRNA, comprising an antisense strand with a complementary sequence to target mRNA and a sense strand, is recognized by the multi-protein RNA-inducing silencing complex, or RISC. The siRNA binds to the RISC, and the strands separate, with the antisense strand remaining bound to the complex.
The activated siRNA-RISC complex recognizes and binds to the complementary sites on the target mRNA. This leads to sequence-specific cleavage of the mRNA by argonaute protein of the RISC complex. The cleaved mRNA is further degraded by the cellular machinery, thereby silencing target gene expression.
