What is the final siRNA concentration to KD a gene in MCF-7 cells? Also share information about transfection reagent for MCF7 cells?

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Mohammad Alzeyadi

Hi..

It is not uncommon for siRNA-mediated knockdown of gene expression to be inefficient or incomplete, especially in certain cell types or under certain conditions. There are several factors that can affect the efficiency of siRNA-mediated gene knockdown, including the quality and design of the siRNA, the cell type and culture conditions, and the transfection reagent being used.

If you are having difficulty achieving efficient knockdown of BLM and SNAI1 expression in MCF7 cells, there are a few things you can try:

Optimize the siRNA design: Make sure that the siRNA sequences you are using are specific to the target genes and have high knockdown efficiency. You can use tools such as siRNA design software or online siRNA libraries to help design effective siRNA sequences.

Optimize the transfection conditions: Experiment with different transfection reagents, siRNA concentrations, and transfection times to find the optimal conditions for your specific cell type and assay.

Consider using other knockdown methods: If siRNA-mediated knockdown is not successful, you might consider using other methods, such as CRISPR/Cas9-mediated gene knockout or gene expression inhibitors, to achieve the desired effect.

I hope this information is helpful!

Mohamed Khashan

Hi Maria Razzaq

24 hours are not enough to get efficient suppression, for some genes that have high to moderate expression level in a certain cell line, siRNA expression can go down not more 15% even after 48 hours. So before taking any other step, transfect your cells with different concentrations of siRNA (in general for 40000 at maximum, a range of 1-50 RNAi duplex is acceptable), test the suppression level after more than 45 hours but not more than 48 of transfection.

Then repeat the transfection from scratch (with the most efficient concentration estimated from the past step) and after less than 48 hours, transfect the cells again and detect the suppression levels after another less than 48 hours. Here, it is supposed to have an efficient suppression. If not, you can try other optimization.

Pls take in your consideration to use not only one sequence of siRNA.

Magdalena Oroń

Hi,

24h might be too short to observe the downregulation of your target protein, especially if it is very stable and highly expressed. Also concentration of siRNA you use seems way too high - more is not always better. I advice you to perform two transfections day by day with a siRNA concentration in a range of 20-40nM, and collect the cell 48h and 72h after the first transfection. If it does not help much mixing two different siRNAs is also to be considered.