I am a new guy in scientific research. These days I am trying to start pre-experiment of transfection.
Protocol summary: 24-wells, HepG2 & Huh-7 cells, 10000 cells/well, FuGENE or LTX reagent, GFP plasmid.
However, the most highest is Huh7 cells with a rate of 50%, under the fluroscence microscopy. I am afraid that such a low transfection efficiency would affect the following tests.
So are there any modification or method to improve the transfection rate?
Thanks.
Hello Dong,
I would highly recommend you have a thorough read through the FuGENE https://www.promega.co.uk/resources/protocols/technical-manuals/101/fugene-hd-transfection-reagent-protocol/ and/or the LTX reagent. These protocols provide key insight into the steps that need to be taken to increase transfection efficiency.
Both of these transfection reagents have an optimisation protocol. Generally speaking, to improve transfection efficiency you have to first of all make sure that your cells are in good health. By this I mean that they are free from infection (such as mycoplasma infection). Furthermore, you have to make sure that the cells are not passaged too many times as it has been noted that transfection efficiency reduces over time. And of course, do make sure that when you split your cells, they are in the proliferative growth phase rather than an over-confluent stationary phase.
Once you have made sure that your cells are healthy you are ready for optimisation. Cell number, amount of GFP plasmid DNA, amount of transfection reagent, the use of antibiotics in your media can all contribute significantly towards transfection efficiency. I would recommend you to start with the recommended amounts in the protocols that I have linked. Following that, optimise your experiment by altering one factor at a time.
All the best,
Salman.
Dear Dong Chen,
Transfection efficiency depends on several factors e.g.:
• Type of Transfection reagent
• Amount of Transfection reagent (see amount of DNA)
• Size of transfected plasmid (bigger plasmids are usually harder to transfect)
• Amount of DNA (more plasmid usually results in a higher transfection efficiency, until a treshold, where DNA is damaging/toxic)
• Ratio of TF-reagent:DNA
• cell type/line
• cell density
• state of the cells (are they still stressed from passaging...)
...
When I want to transfect plasmids or cell lines I have never transfected I test 2 densities, 2 ratios of reagent:plasmid, 2 amounts of DNA (--> therefore amount of TF-reagent) on day1 and day2 after passaging.
In the best case you get optimal parameters, worst case you see which parameter has which influence and can adapt.
This can be adapted to your time and material capabilities. I definitely suggest reading the reagent protocol for guidelines.
Best,
Felix
Hi Dong,
Felix has good points.
You also have to known if this this is an integratable plasmids or a transient transfection. If is integratable then you can select out all non transfected cells. At some point transfection efficiency should be absolute. There will always be differences between cells in respect to GFP fluorescence depending on how many copies of plasmid integrated per cell.
Good luck,
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