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High-throughput parallel transfection techniques are a prerequisite to performing large-scale experiments, such as genome-wide RNAi screens, in mammalian tissue culture cells. A variety of automated precision devices based on reverse transfection, cell microarray, improved electroporation, and other theories have been developed, which can efficiently and high-throughput transfection of nucleic acid molecules into target cells for clinical research and clinical applications.
Reverse transfection differs from the traditional transfection method in that the traditional method requires inoculating the plate one day in advance, incubating the cells for 24 hours, and then adding the transfection complex to transfect the cells at a certain confluence level; whereas reverse transfection does not require inoculation of cells in advance, but first adds the transfection reagent and pre-transfected nucleic acid mixture on the plate and incubates them together. The cells are then added to the plate and cultured. The advantage of this method is not only that it can save a lot of time, but also that the transfection efficiency is high. The reason for this may be due to the greater surface area of the nucleic acid complex in contact with the transfection reagent when the cells are not yet attached to the wall, making transfection more efficient. Numerous experiments on cell lines have shown that reverse transfection gives better RNA interference than conventional transfection methods, even for some difficult-to-transfect cell lines such as HepG2 cells. In the reverse transfection experiments, the density of cells no longer bothered the experimenters, and some experiments showed that the cell density at the time of harvesting had no significant effect on the RNAi results, while the trypsin treatment of the harvested cells and the mixing of the suspended cells and the transfection complex before spreading the plate culture not only saved one day of culture time than the traditional method but also did not need to consider the problem of cell density. The concept of reverse transfection is suitable for the needs of high-throughput transfection, so that transfection is no longer confined to manual operation, and can be simply automated to realize efficient and large-scale experimental work.
In large-scale experiments, transfections are typically performed in 96- or 384-well plates using liquid-handling robotics. Reverse transfection of cells on plasmid or siRNA arrays is a powerful alternative method to perform high-throughput transfections for phenotypic data acquisition by light microscopy.
One of the limitations of reverse transfection is that the application of different cell lines so far requires different protocols for making siRNA or plasmid arrays, which involves a significant amount of development and testing of the technique. Another key aspect is the potential for cross-contamination of the array sites as point density increases; Therefore, the optimization of the array layout is very important.
Microfluidics is the study of techniques that can handle small amounts of fluid by using tiny channels (typically tens to hundreds of microns) in tiny sizes.
Cell transfection can be carried out in two microfluidic ways. The first is to use microfluidic technology to generate microdroplets as carriers to dissolve the mixed gene fragments and deliver them to the target cells. The second is to manipulate microfluidic encapsulated cells and use electroporation for transfection. This effectively avoids the complex process of conventional electroporation and allows precise manipulation of cells. An additional advantage is that controlling the droplets of encapsulated cells is easier and more precise than with individual cells in an aqueous solution, minimizing electrochemical reactions at the electrodes.
Microfluidics devices and platforms (including microfluidic chips and microarrays) for gene delivery have been extensively reported, with the advantages of high throughput and low reagent consumption, and independent control of liquid conditions, but most research has focused on chemical transfection or electroporation.