Transfection reagent selection

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Selection of transfection reagent is sometimes a puzzling issue. Frequently labs try stay with their traditional technology and do not want to change it due to "optimisation" issue.

This was a case in our lab. For years we used Lipofectamine reagent (Invitrogen) for transfecting our "easy-to-transfect" cells like CHO-K1, HeLa, NIH-3T3, HEK293. The same reagent was used also for less easy to transfect cells like HepG2. The results were generally acceptable regarding reporter gene assay and expression of recombinant proteins. The transfection reagent was relatively cheap since Invitrogen gave us a nice discount.

The protocol of transfection using Lipofectamine reagent is relatively simeple. Prepare liposome complexes, add them to your cells, add serum-containing medium after 5 h and change medium after 24 h.

Recently we got major problems with 2 tagged proteins we wanted to express in eukaryotic cells. Students spent 3-4 months trying to express the proteins in various cell lines controlling the efficiency of transfection, optimising the plasmid construct and so one. Everything was done with Lipofectamine reagent.

After giving up all other possibilities the change of transfection reagent was proposed. Since there are hundreds of various transfection reagents on the market the choice was done according to the reputation of the company, possible discount and availability of a free sample. We decided to give a try to FuGENE HD from Roche.

First of all the students were happy with the transfection protocol. Its a typical "pipet-and-forget" protocol. Prepare liposomes, pipet them to the cells and harvest after 2-3 days. No 5 h waiting, no 24 h medium change.

After the first try (parallel experiment with Lipofectamine and FuGENE HD) with GFP transfection it became obvious that we get much better efficiency (number of transfected cells) when FuGENE HD is used. But obviously it was not all. The very first try with our complicated tagged protein constructs reveraled wonderful expression of recombinant protein from all plasmids we cloned and in all cells we analyzed.

This story actually confirms that sometimes it is worthwhile to try somethiing new.

Conditions for transfection of HepG2 cells.

  • Use 1 µg plasmid and 2.5 µl FuGENE HD for 1 ml culture medium (for 1 well in 24 well plate)
  1. On the day before transfection seed cells at 60% confluency using 900µl culture medium per well.
  2. In a cell culture 96 plate prepare 100 µl culture medium containing 1µg plasmid.
  3. Add 2.5 µl FuGENE HD to 100 µl culture medium, containing plasmid. Pipet FuGENE HD directly into medium not on the wall of the well.
  4. Mix content by rotating the plate (do NOT pipet up and down).
  5. Incubate 15 min at RT.
  6. Transfer plsmid/FuGENE mix to the cells.

You can harvest your cells 48 h after transfection. But usually it is a good idea to check for the optimal duration of the experiment.


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