I have been trying to silence a gene of interest but I could not get silencing efficiencies beyond 50%
My method of detection is via RT-qPCR and samples were extracted 48hrs post transfection. Are there such genes that are so difficult to silence?
Here are the materials i used:
siRNA (Concentration used)
1st siRNA: Dharmacon smart pool ONTARGET (5, 10, 20nM)
2nd siRNA: Ambion siRNA 2 sequences (5, 10nM)
Reagent:
oligofectamine
optimem
Cell lines:
MDA-MB-231
BT549
MCF7
Hello,
Lipofectamine RNAiMAX should work slightly better. How many siRNAs are you transfecting simultaneously? I suggest to try them all separately if you have them...choose the best one and then do a tritation (5-50nm).
The main difference with RNAiMAX is that oligofectamine is not serum compatible while lipofectamine is.
If you are checking mRNA, could be OK to prepare the RNA extraction 24h post transfection (rather than 48h).
Also make sure cells are 50-70% confluent when you start the transfection.
Best Regards
Hi,
check if the gene is essential or not (http://www.essentialgene.org/). If yes, all the cells without sufficient gene expression will die, leaving only the cells with insufficient silencing still alive. If it is not an essential gene, consider ordering new siRNAs or try CRISPR.
Best wishes.
Check the protein level expression by WB/Immunofluorescence the number of transcripts detected by RT-PCR not always directly correlates with protein expression especially when siRNA is used and not Cas9 knockout. So you actually might have successful silencing but RT-PCR would still show the substantial number of the mRNA for the target present in the cells. I also agree with the possibility suggested by Koraljka Husnjak
I agree with Koraljka Husnjak , you could be hitting an essential gene. One further way to check would be to evaluate cell death after KD vs. control. If it's just low efficiency you can use your fast growing cell lines for singe cell clones in 96 wells (either by limiting dilution or 0.5 cells/well) and thus get "pure" monoclonal populations.
Hope that helps
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line