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Basic questions

siRNA Basics transfections

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#1 madelingirly

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Posted 07 December 2013 - 03:55 PM

Dear all,

I want to silence a specific gene using siRNA and I never do such experiment before, so I have some basic questions.

1) about transfection and gene silencing control.

After I finish tranfection procedure, I have normal 1ry AB against expressed protein so can I check the gene silencing using this kind of AB.

2) After transfection and make sure that my gene is knocked down, I need to do experiment by 1st trypsinonized the cells, remove it and recultrue it again on my specific experiment conditions (this is my procedure). so each time I do experiments I need to make the transfection experiment first?? I cant have stock of silenced cells, or reculture it in plates and take cells every time I need.

3) do u suggest any comprehensible review or article or book for more information regarding siRNA practical steps

 

Thank u

Madelin

 



#2 pcrman

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Posted 08 December 2013 - 09:29 AM

Hi Madelin,

1) about transfection and gene silencing control. After I finish tranfection procedure, I have normal 1ry AB against expressed protein so can I check the gene silencing using this kind of AB.

->As long as your AB is still working. You may also check mRNA expression by RT-PCR.

 

2) After transfection and make sure that my gene is knocked down, I need to do experiment by 1st trypsinonized the cells, remove it and recultrue it again on my specific experiment conditions (this is my procedure). so each time I do experiments I need to make the transfection experiment first?? I cant have stock of silenced cells, or reculture it in plates and take cells every time I need.

->Please keep in mind that after siRNA transfection, RNAi effect lasts only for a week or so. Once you pass the cells, you can lose RNAi activity in the cells. If you want stable knockdown, you need to construct the siRNA as shRNA vector and use the vector to transfect cells. 

 

3) do u suggest any comprehensible review or article or book for more information regarding siRNA practical steps

->If you have never done RNAi, yes, read some protocols or guides to familiarize yourself with the technique. Here are a few:

 

http://www.promega.c...a-interference/

http://www.biotechni...ques-45525.html



#3 madelingirly

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Posted 12 December 2013 - 09:58 PM

Dear 

pcrman

Thanks so much for ur valuable comments

Madelin







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