Applications: High Throughput Transfection

High Throughput Transfection

Advances in high throughput (HT) screening as well as availability of multi-well cDNA/shRNA/siRNA/miRNA libraries have made high throughput transfection of different nucleic acids commonplace. High throughput transfections can be carried out in multi-well formats (such as 96-well, 384-well, etc.) using reverse transfection protocols that are amenable to automated robotic systems.

JANUS® Automated Workstation from PerkinElmer

Forward Transfection

The most routinely employed transfection protocol where cells are seeded a day prior to transfection is referred to as "forward transfection". Forward transfection methods work well for most adherent cell types that are seeded a day prior to transfection in order to achieve an actively dividing cell population at the time of transfection. A typical forward transfection protocol using TransIT-X2® Dynamic Delivery System can be found here.

Reverse Transfection

For high throughput applications, a "reverse transfection" protocol, where freshly passaged cells are added to pre-plated transfection complexes is ideal as it reduces overall experimental time for the end user. Cell culture time can be further reduced by using frozen assay-ready cells for some experiments (see example here). Reverse transfections are also compatible with most automated robotic systems.

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Typical Reverse Transfection Protocol Workflow. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward transfection protocol. This reduces hands-on time for the end-user by one day compared to forward transfection and makes the protocol amenable to liquid handling.

There are also a few variations of standard reverse transfection protocols that are employed by researchers; brief explanations are included below:

Modified Reverse Transfection

In "modified reverse" transfections, cells are passaged and plated immediately before transfection complexes are added to the cells. In this scenario, adherent cells are loosely adhered to the plate surface by the time they interact with the transfection complexes.

Solid Phase Reverse Transfection

If the nucleic acid to be transfected is immobilized or spotted in a multi-well format already as in the case of cDNA/shRNA/siRNA screens, the transfection protocol is referred to as "solid phase reverse transfection".

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