I would like to know the optimum confluency of cells at the time of transfection. I have seen some protocols that suggest 30-50% and some 50-80%. So, I am confused about the confluency to start the experiment. I am using SKOV3 cell line.
Please correct me if I am wrong, if I am increasing the concentration of siRNA accordingly I should also increase the concentration of my transfection reagent. But the transfection reagents are so expensive, so I am planning to try using Polyethylene imine as transfection reagent. Has anyone used PEI for transfecting siRNA?
Hai Bhavani,
not all tfx reagents are so expensive. We compared the most used, see the attachés link.
however if you look into the cost the cellculturing is more costly then the reagent. If you are using a tfo reagent inducing slot of celldeath the experiment Will even more costly.
Do not forget the cost of the siRNA. If the delivery efficacy of the tfo reagent is low you Will increase the cost again.
Depending on the assay you are performance after downregulation you need cells which are not effecten by inwandelde Side effects.
in general you would like a reagent with high delivery efficacy, low cell toxicity and no side effects.
http://www.synvolux.nl/sirna-transfection-reagent/sirna-transfection-reagent-comparison
http://www.synvolux.nl/sirna-transfection-reagent/cost-effective-sirna-transfection-reagent
Bhavani,
The issue of confluency is complicated and depends on the target and cell.
In general you would like to measure in health growing cell i presume.
If the read-out is performed on the protein, be aware of the halflifetime of the protein.
A halflifetime of 2 days means in your assay, performed at 48h, that the value is still 50% if you would be able to hit all mRNA. The question then , is your protein assay sensitive enough to see the difference.
Second issue what is the halflifetime of the siRNA, this is cell depending and might be shorter in fast growing cells.
So with a protein halflifetime of 1 day and a celldouble time of 16 hours you might perform the readout after 2 days. You need to start with a confluency of 12,5%.
If the readout is performed with RT-PCR it will be more easy but be aware that you read-out is to be measured in growing cells, depending on your research question.
Bhavani, This does not address your question in full but I thought I would point out that the ICCB-Longwood Screening Facility at Harvard Med School has a list online of a variety of reagents appropriate for siRNA transfection. I hope this is a useful supplement to the other answers that have been posted. Best, Stephanie
http://iccb.med.harvard.edu/rnai-lipid-panel-2013/
I have used Dharmafect I successfully in megakaryocytic cells. I highly recommend it for use with naked siRNA. To keep costs down, you can use the same concentration of Dharmafect for a narrow range of siRNA. For example, I used 5 microliters for both 50 and 100 nM siRNA. Good luck, Steve
Thank you everyone for your detailed response and your helpful suggestions.
@ Marcel : thank you for your detailed response.I dd not consider the halflife of the protein,so thanks for suggesting .I am using SKOV3 cells and trying to knockdown STAT3.The doubling time for SKOV3 is 48hrs.I do Western Blot to check for the knockdown and i couldnt see a very efficient knockdown while comparing the control and siRNA transfection. So I am trying to figure out the cell confluency at the time of transfection to increase the chances of knockdown.
@Andrei: thanks for the suggestion,sure I will try electroporation.
@ Stephanie :Thank you Stephanie for attaching an useful link for the transfection reagents.Its a great help.
@Chapel :Thank you Chapel, i was worried about increasing the siRNA concentrations as I might use up the transfection reagents ,but after seeing your comment I am planning to use the same volume of transfection reagent for marginally higher concentration of siRNA.I have been using Lipofectamine RNAimax.
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