Transfecting HMEC
Human Mammary Epithelial Cells are naturally non-immortal cells commonly used in mechanistic studies to understand growth, aging and senescence in relation to malignant cells. Studies using these cells have given insight into cellular pathways of aging and the in vivo development of malignant cell types. These results are paving the way for therapeutic interventions for breast carcinogenesis. With the lack of senescence playing a major role in the immortalization of HMEC cells, understanding pathway checkpoints that circumvent senescent barriers will be key in understanding the development of tumorigenesis.
HMEC Cell Transfection Protocol
A pre-optimized HMEC Transfection Kit is available from Altogen Biosystems, which includes:
- HMEC Transfection Reagent (0.5 ml / 1.5ml / 8.0 ml)
- Transfection Enhancer (0.5 ml), and
- Complex Condenser (0.5ml)
The kit is optimized for transfection of DNA and RNA into HMEC cells following either a standard or reverse transfection. The optimized protocol for a 24-well plate to transfect HMEC cells is described below:
- Plate 7,500-12,000 HMEC cells per well in 0.5 ml of complete growth medium 12-24 hours prior to transfection
- Wash with 1xPBS and add 0.5 ml of fresh growth medium
- Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and (referred to a final volume including growth medium)
- 750 ng DNA (or mRNA), or
- 30 nM – 50 nM of siRNA (or microRNA)
- Incubate transfection complexes at RT for 15-30 minutes
- Optional: Add 2 µl of Complex Condenser. This reagent increases the transfection efficiency by reduces the size of transfection complex; however, it may increase cell toxicity
- Add prepared transfection complexes to 0.5 ml of complete growth medium with HMEC cells (from step 2)
- Incubate cells at 37ºC in a humidified CO2 incubator
- Assay for phenotype or target gene expression 48-72 hours after transfection
Optional: Adding Transfection Enhancer reagent can increase transfection efficiency. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection
If the viability of HMEC cells being transfected is affected at 16-24 hours post-transfection, changing the growth medium and eliminating redundant exposure of cells to transfectant can reduce the level of cytotoxicity
Figure: siRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into HMEC cells. At 48 hrs post-transfection the cells were analyzed by qPCR for gene expression levels and normalized to 18S rRNA levels. The data is represented as expression levels relative to the untreated sample. Data are means ± SD (n=3).
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