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Low Transfection Efficiency? 5 Points You May Not Have Considered

Updated: Aug 28, 2020


We Are Cellix Low transfection efficiency

All researchers working with cell transfection methods, whether in academia or in industry, know about the pains involved in optimising cell transfection. Sometimes some cells just seem to be more resistant to transfection than others, often for frustratingly vague reasons.

So we've put together a 5 point list covering the major factors impacting transfection efficiency. Use these points as a guide to help you start to optimise your experiments and get more transfected cells.

Is poor transfection efficiency wasting time in your high throughput processes? We can help. Get in touch.

The 5 major factors that affect transfection efficiency are

1. Cell viability

2. Cell type

3. Transfection Methods used

4. Confluency and replication state

5. Nature of insert or expressed protein

1. Cell Viability

When considering the transfection process as a whole, transfection efficiency and cell viability are intrinsically linked - there's little point in having high transfection efficiency no cells survive.

Prior to transfection, cells should be >90% viable and been given time to recover from passaging.

Factors impacting viability are manifold; if you're seeing low viability of cells prior to transfection, check the following:

Nutrition: that the media, serum and supplementation requirements are the right ones for your cell line. Each cell line has its own unique requirements.

Contamination: Contamination can be biological or chemical. Biological contamination of cell lines by bacteria, viruses, mould, yeast, and mycoplasma can occur and impact negatively on the health of your cell. With bacteria, a sudden decrease in pH is often seen; with yeast and mould the pH rises in the later stages of infection. Other warning signs include a turbid cell culture or pronounced cell death. Biological contamination can be prevented by good aseptic technique.

Cross-contamination by other mammalian cell cultures is a widespread and under-diagnosed issue - a study in 1976 tested 246 cell lines and found that a stunning 30% of these were incorrectly labelled. If you're uncertain, look at isotyping, DNA fingerprinting and karyotyping of cells to diagnose the problem.