This article describes a method for reverse transfection of siRNA into non-adherent adipocytes. The process involves preparing a suspension of siRNAs, forming transfection complexes, and delivering siRNA into the cytoplasm to silence target gene expression.
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.
Pipette the siRNA with improved minimum essential medium to mix, and incubate for 5 minutes at room temperature. Add the transfection reagent and the improved minimal essential medium to the siRNA, and pipette to mix. Incubate it for 20 minutes at room temperature. Then, add the transfection mix to each well of the collagen-coated plate.
Wash the cells in the 100-millimeter Petri dish twice with DPBS. Add 5X trypsin to the cells, making sure to cover the entire surface with the trypsin. Wait for 30 seconds, and carefully remove the trypsin. Incubate the Petri dish for 5 to 10 minutes at 37 ℃ in the incubator.
Then, tap the dish to detach the cells, avoiding cell damage. Add 10 milliliters of DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin, to neutralize the trypsin. Carefully pipette the medium up and down to detach the cells and homogenize the cell suspension.
Count the cells using a Malassez counting chamber or an automated cell counter, and adjust the concentration of the cells to 6.25 x 105 cells/mL of medium. Seed 800 microliters of the cell suspension per well of a 12-well plate, or 400 microliters of the cell suspension per well of a 24-well plate containing the transfection mix.
Incubate the plates in a cell culture incubator, and do not disturb them for 24 hours. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin, and return the cells to the incubator.
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