Reverse Transfection


Transfection is a method to transfer genetic material into cells in order to add or alter the expression of a specific gene in a cell. Transfection experiments are conducted in order to study gene expression, cell processes, DNA and RNA structure, function, and coding sequences.  Transfection is typically performed using transfection reagents to transfer plasmid DNA, siRNA or miRNA into a cell.

Transfection reactions may be accomplished through the use of commercially available reagents, as well as mechanical methods of introducing DNA into eukaryotic cells.  Reverse transfection is defined as the process of adding the encapsulated cargo molecule (plasmid DNA, siRNA, miRNA) while simultaneously plating the cells.

An alternative definition of reverse transfection refers to cell-based array experiments in which cells are pipetted onto pre-plated cargo molecules.  Such approaches are often used for large scale RNAi screening studies, where libraries of cargo molecules are on a slide, dish or plate.  The array is then incubated with cell cultures such that the cells become transfected.  One of the most common applications utilizing array-based reverse transfection is large scale siRNA (or large scale microRNA) library screening using siRNA reverse transfection.  Success or failure of reverse transfection depends on quality of cells, experience of the researcher, and methods used in the reverse transfection.

The micro-array based reverse transfection method was invented in 2001 by Junald Ziauddin and David M. Sabatini.  Using gelatin and nucleotides (DNA or RNA), the mixture is printed onto a non-porous surface, such as glass.  After drying, cultured cells are added on top of the printed nucleotides, limiting inherent variation in handling large amounts of oligonucleotides and enabling high throughput screening experiments to be performed on the same type of cells.