Biochemistry

scientificprotocols authored over 3 years ago

The term reverse transfection comes from the invention and development of a microarray-driven gene expression system by Junald Ziauddin and David M. Sabatini in 2001. As DNA are printed on a glass slide for transfection process to occur before the addition of adherent cells, the order of addition of DNA and adherent cells is a reverse of that of conventional transfection. Hence the word “reverse” is used.

Reverse Transfection Process

Preparation of transfection mix for printing onto a slide

DNA-gelatin mixture can be used for printing onto a slide: Gelatin powder is first dissolved in sterile MilliQ water to form 0.2% gelatin solution. Purified DNA plasmid is then mixed with gelatin solution and the final gelatin concentration is kept greater than 0.17%. Besides the use of gelatin, atelocollagen and fibronetin are also successful transfection vectors for introducing foreign DNA into the cell nucleus.

Printing of DNA-gelatin mixture onto a slide

After the DNA-gelatin mixture preparation, the mixture is pipetted onto a slide surface and then the slide is put into a petri dish with a cover. A drying chemical is added into the dish to dry up the solution. Finally, cultured cells are poured into the dish for plasmid uptake. However, with the invention of different types of microarray printing system, hundreds of transfection mixes containing different DNA of interest can be printed on the same slide for the uptake of plasmids by cells. There are 2 major kinds of microarray printing systems manufactured by different companies: Contact and non-contact printing system.

An example of non-contact printing system is Piezorray Flexible Non-contact Microarraying System. It uses pressure control and a piezoelectric collar to squeeze out consistent drops of approximately 333 pL volume. The PiezoTip dispensers do not contact the surface to which the sample is dispensed, thus contamination potential is reduced and the risk of disrupting the target surface is eliminated. An examples of contact printing system is SpotArray 72 (Perkin Elmer Life Sciences, Inc) contact spotting system. Its printhead can accommodate up to 48 pins and creates compact arrays by selectively raising and lowering subsets of pins during printing. After printing, the pins are washed with a powerful pressure-jet pin washer and then vacuum-dried, eliminating carryover. Another example of contact printing system is Qarray system (Genetix Inc). It has three types of printing systems, QArray Mini, QArray 2 and QArray Max.

After printing, the solution is allowed to dry up and the DNA-gelatin is sticked tightly at the position on the array.

Use of HybridWell for reverse transfection of gelatin-DNA into cells on a slide

Firstly, the adhesive from the HybriWell is peeled off and then attach the HybriWell over the area of the slide printed with the gelatin-DNA solution. Secondly, pipette 200ul of transfection mix into one of the ports of HybriWell. The mix will distribute evenly over the array. Incubate the array at particular temperature and time length depending on the types of cells used. Thirdly, pipette away the transfection mix and pull off the HybriWell using a thin tipped forceps. Fourthly, put the printed slide treated with transfection reagent into a square dish with the printed-side facing up. Fifthly, gently pour the harvested cells onto the slides (don’t pour directly on the printed areas). Finally, place the dish in a 37oC, 5% CO2 humidified incubator and incubate overnight.

Other transfection reagents for reverse transfection

Effectene Reagent is used in conjunction with the Enhancer and the DNA condensation buffer (Buffer EC) to achieve high transfection efficiencies. In the first step of Effectene–DNA complex formation, the DNA is condensed by interaction with the Enhancer in a defined buffer system. Effectene Reagent is then added to the condensed DNA to produce condensed Effectene–DNA complexes. The Effectene–DNA complexes are mixed with medium and directly added to the cells. Effectene Reagent spontaneously forms micelle structures that show no size or batch variation, as found with preformulated liposome reagents. This unique feature ensures excellent reproducibility of transfection complex formation. The process of highly condensing DNA molecules and then coating them with Effectene Reagent is a particularly effective way to transfer DNA into eukaryotic cells.

Advantages and disadvantages of reverse transfection techniques

The advantages of reverse transfection than conventional transfection are: Firstly, the addition and attachment of target cells to the DNA-loaded surface can lead to higher probability of cell-DNA contact, potentially leading to higher transfection efficiencies. Secondly, less labour-intensive and saving materials. Less DNA is required for the success of transfection. Thirdly, high throughput screening: each time hundreds of genes can be expressed in cells together on a single microarray for studying gene expression and regulation. Fourthly, parallel cell seeding in a single chamber for 384 experiments together with no physical separation between experiments increases the screening data quality. Well-to-well variations occur in experiments done in multiwall dishes. Fifthly, exact replicate arrays can be produced as same sample source plate can be dried and printed on different slides for storage at least 15months without apparent loss of transfection efficiency.

There is a disadvantage of using reverse transfection: Reverse transfection is more expensive because highly accurate and efficient microarray printing system is needed to print the DNA-gelatin solution onto the slides. Firstly, applications with different cell lines have so far required variations in the protocols to manufacture siRNA or plasmid arrays, which involves a considerable amount of development and testing. Secondly, possibility of cross-contamination of the array spots when spot densities increase; therefore, optimization of the array layout is important.

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