Asked 25th Mar, 2015
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Why are my cells dying?

I've been having this problem for the past week now and I don't know what to do anymore. Following siRNA transfection a week ago, all cells I plated on coverslips died. I felt it was because I used a high volume of transfection reagent (But that has never been the case). So, I decided to seed a new batch onto coverslips and the same problem occurred after 24 hours. Thus, I decided to recover a new vial of low passage cells from liquid nitrogen. All cells behaved well and I went ahead to seed them onto coverslips once again. However, following siRNA transfection 24 hours ago, the cells underwent massive death once again. I'm totally confused as I've never encountered this before. What could I have done wrong. 

Most recent answer

Ma Jun
Binzhou Medical University
Have you considered that maybe your cell line is so sensitive to this siRNA that knocking down this gene will make cells dead? Why not lower the dose of siRNA seeded on your plate, and then see what happen.  

All Answers (8)

Henrik Helmfors
Stockholm University
Are you knocking down the same gene as before? I have seen my cells dying when trying to knockdown genes that are important (for nutrient uptake). Also some transfection reagents are a little toxic.
Yes, same gene.
Richard Marcotte
National Research Council Canada, Montreal, Canada
Have you plated these cells on cover slip before? Cover slip and cell culture plate are not equivalent (these are specially coated for cell culture). Some cell lines don't attach/survive on cover slip.
Piyush Jain
University of Florida
Please provide more information on cell lines you are using. Are you seeing any contamination? As Richard mentioned, they might not like cover slips. I had issues growing some macrophage cell lines on cover slips. Do some controls and let us know how it looks. Compare them on a plate and have both lipofectamine treated and untreated cells. Also if possible compare them with scrambled or control siRNA wells. Good luck!
Thank you, Piyush. I've checked the cells (MCF7s) for possible contamination and they came out clean. 
Yunfu Lin
University of Texas MD Anderson Cancer Center
If the reagent is excluded to be a possible issue, I would suggest you to plate the cells on cover slips such as 24 hours (or even longer) before siRNA transfection.
Breanna Symmes
University of Colorado
Is the problem only on the slides, or are you seeing decreased growth in your plates, as well? If you see decreased growth in all of your cultures, you may have a mycoplasma issue or another small, hard-to-detect contaminant.
Ma Jun
Binzhou Medical University
Have you considered that maybe your cell line is so sensitive to this siRNA that knocking down this gene will make cells dead? Why not lower the dose of siRNA seeded on your plate, and then see what happen.  

Similar questions and discussions

How can I perform SiRNA concentration scaling? by nM or pmol per num of cell / surface area?
6 answers
  • Martin FordeMartin Forde
I'm looking to scale up my siRNA reverse transfections from a 6-well to 384-well plate for adherent SH-SY5Y. Should I be scaling the siRNA concentration by  surface area in the well, or by the total volume of media in the well?
For background, I have settled on 42pmol in a 6well with 2ml of media on a 9.5cm^2 surface area. That's a siRNA concentration of 21nM. this gives me consistently 50% KO (+/-) 3% SE for all of my targets. It suits our purposes very well, and follows a predictable dose response curve, so i know that the effect is real and that my siRNAs are targeting properly.
When I scale to 384-well, the total volume in each well will be 50ul for on a surface area of 0.056cm^2. So do I scale down my siRNA concentration by nM concentration or surface area?
Lipofectamine / RNAiMAX method appears to work by surface area:
By surface area = 42pmol / (9.5cm^2 / .056cm^2) = approximately 0.25pmol per 384 well
I'm concerned because I'm working with the Viromer Green transfection reagent, and their protocol scales off of nM concentrations. But if I follow this reasoning and suspend 0.25pmol of siRNA in 50ul of media I get a siRNA concentration of 5nM. If I scale off of nM like Viromer suggests, then I would need to plate:
21nM in 50ul media = 42pmol / (2000ul / 50ul) = 1.05pmol per 384-well.
I just think that 1pmol per 384-well seems really high. Am I wrong to be concerned? What would you suggest?

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