Transfection Information

Research with HeLa Cells

Being a vicious cancer cell in its own right, the HeLa cell line was originally derived for use in cancer research. HeLa cells are extremely malignant, even when compared to other cancer cells from malignant sources. Telomere shortening, which is implicated in eventual cell death after a number of divisions, is prevented in HeLa by the enzyme telomerase which restores telomeres during mitosis. This is why HeLa cells are considered immortal and do not experience cell death after a set number of cell divisions like normal cells other cancer cells.

Even though HeLa cells are very hardy, the HeLa cell line is fairly easy to transfect and very receptive to viral vectors, making it a popular research tool in virology. Since their initiation, HeLa cells have been used in transfection experiments all over the world to study viruses. It was the HeLa cell line that took up the polio virus and made the vaccine that eventually eradicated polio. HeLa is also receptive to Human adenovirus 3, Encephalomyocarditis virus and Human polioviruses 1, 2, and 3.

HeLa Cell Transfection Protocol

A pre-optimized HeLa cell line transfection reagent is available from Altogen Biosystems – HeLa Transfection Kit.  The kit is optimized to transfect miRNA, siRNA or DNA plasmid following either forward or reverse transfection protocols. Below is the optimized protocol to transfect a 24-well plate of HeLa cells:

  1. Prepare HeLa cell suspension as follows:
    • Trypsinize cells for at 37°C for 5 minutes
    • Count cells and dilute cell suspension in complete growth medium to 5 x 104 cells/mL
  2. Prepare the transfection complexes by adding 40 µL of serum-free medium, 5.5 µL transfection reagent and one of the following in a tube:
    • 750 ng plasmid DNA
    • 30 nM – 50 nM of miRNA or siRNA
  3. Incubate at RT for 15 – 30 minutes for allow complexes to form
  4. Optional: Adding 2 µl of the Complex Condenser reduces the size of transfection complexes and increasing transfection efficiency; however, cell toxicity may be increased
  5. Plate 15,000 – 25,000 cells per well in a total of 0.5 mL complete growth medium
  6. Add prepared transfection complexes (from step 3 or 4) to the plate
  7. Incubate plate at 37ºC in a humidified CO2 incubator
  8. After 48 – 72 hours, assay the plate for the phenotype or target gene expression of interest

Figure. HeLa cells were transfected with CyclophilinB siRNA and quantified by qRT-PCR following the recommended transfection protocol. CyclophilinB mRNA expression levels were measured 48 hours post-transfection. 18S rRNA levels were used as a housekeeping gene to normalize the CyclophilinB data. Values are normalized to the non-treated sample and data are presented as means ± SD (n=3).

Transfection Controls

Three controls should be included on every transfection plate: a negative control, positive control and non-treated control.

Positive Controls

A positive control ensures the system is working and delivery conditions are optimal.  Results of the positive control helps in understanding any day to day experimental differences.  Positive controls consists of these characteristics:

  • Gene is a housekeeping gene: Ensures target gene is abundantly expressed in all cells and at constant levels
  • Abundant expression: Enables easy quantitation of gene expression mRNA or protein levels
  • Non-lethal: Altering expression levels of the positive control should not affect phenotype or viability
  • Common targets: CyclophilinB (PPIB), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin (LMNA)

Negative Controls

Negative controls have no known derived target and are commonly called a “scrambled” control.  The ideal negative control mimics the test compound in G/C content, length and contains identical base modifications.  Negative control samples are analyzed the same as test article samples.  Negative controls exhibit these characteristics:

  • Gene targets: Alien sequences are used (no known target) so that it does not alter expression levels of mRNA or protein
  • Non-lethal: Does not affect viability or global gene expression from non-target design

Non-Treated Controls

Non-treated and mock transfection controls (i.e. transfection reagent only) are also essential.  The mock and negative controls help to understand experimental toxicity when expression data is compared to the non-treated samples.


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