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non-silencing shRNA control


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#1 Jeannine

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Posted 08 April 2010 - 05:57 PM

Hi!

I'm doing a lot of retroviral transductions with shRNAs at the moment and was just wondering if I have to transfect a non-silencing shRNA everytime I do a transfection or if I can just use my stable cell line expressing the non-silencing shRNA from the first experiment.
Someone told me that I can only use a ns shRNA cell line as a control if I generated it at exactly the same time as the knockdown cell line...
Do I need 10 different control cell lines for each transduction or one for all???

Hope you can help me,
Jeannine

#2 Functional Screens

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Posted 08 April 2010 - 08:44 PM

You better transduce your model cell line(s) with specific and control shRNAs side by side each time.

#3 than4

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Posted 08 April 2010 - 09:06 PM

I'm only doing transient knockdowns but I include a non-silencing control (amongst others) every time I do a transfection.

I would imagine with shRNA where you are generating a stable cell line that you need to generate a non-silencing cell line at the time you do the transfections to generate the knockdown cell line. However, if you are knocking down a variety of different genes in the same cell line then I don't know. Because you would use the same non-silencing shRNA as previously and you already know it doesn't have any effects on your new gene of interest.

I read somewhere that your non-silencing control should be the sequence used for silencing but with the letters mixed up - not sure how accurate this statement is either.

#4 Jeannine

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Posted 08 April 2010 - 10:25 PM

Hi,

Thanks for the help.
I'm using the same cell line for all my transductions and the same control shRNA, and therefore thought one control cell line for all future transductions would do. So every time I do a retroviral transduction I have to generate a stable control cell line, which means Puromycin treatment for 1-2 weeks, picking clones, testing them.... that seems a lot of work for a control...

Jeannine




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