6 replies to this topic

[banou]

I am trying to knockdown dicer (by a plasmid which encodes the siRNA against dicer and is resistance to Puromycin) to see any possible regulation of my gene of interest by miRNA. I have found that the optimum puromycin concentartion for SW620 cells are 0.5ug/ml thus The cells are transfected ( with three different dicer siRNA olasmids) using Fugene HD, 24h prior to antibiotic treatment. they are under treatment for 48h. then the media is replaced with fresh DMEM and 24h later the cells are harvested for RNA isolation. I have two problems: first: at the end I have afew cells for RNA isolation.
2nd: the taqman results doesn't show any dicer down regulation and it's expression is almost as much as scrambled one.
I appreciate any comment.

[Functional Screens]

I tried to knockdown Dicer with siRNA, vector-shRNA, retroviral-shRNA in 2002.  The best knockdown I can get is around 60%.  Please note shRNA requires Dicer to process to siRNA, therefore you might not be able to get >70% knockdown.
It may be lethal especially in embryonic cells.
In your case, transfection efficiency matters.  Linerization of vector is highly recommended.  Longer selection time may be needed for vector integration.

[banou]

Functional Screens, on Apr 21 2010, 07:10 PM, said:

I tried to knockdown Dicer with siRNA, vector-shRNA, retroviral-shRNA in 2002.  The best knockdown I can get is around 60%.  Please note shRNA requires Dicer to process to siRNA, therefore you might not be able to get >70% knockdown.
It may be lethal especially in embryonic cells.
In your case, transfection efficiency matters.  Linerization of vector is highly recommended.  Longer selection time may be needed for vector integration.

[size="2"]Dear Functional screens,
In case of transfection effieciency, since the cells are treated with puromycin, all of live cells supposed to have the shRNA other wise they will die under selection.so I assume that almost 90% of my cells are transfected (according to results from  GFP transfection in parralel with shRNA).
you said it's better to linearize the plasmid before transfection, If I understood properly, you mean cutting the plasmid with an enzyme, opening it then transfection.

[Functional Screens]

banou, on Apr 21 2010, 11:28 PM, said:

[size="2"]you said it's better to linearize the plasmid before transfection, If I understood properly, you mean cutting the plasmid with an enzyme, opening it then transfection.

Non-homologous end-joining will join many copies of linearized vector and then homologous recombination will integrate multiple copies of your vector (the drug selection gene and shRNA expression cassette) into host genome.  For example, a (unique) ScaI site in the Amp gene can be used to linearize the vector.  Also, you might need to do longer selection to ensure the recombination/integration.

[banou]

Functional Screens, on Apr 22 2010, 04:25 PM, said:

banou, on Apr 21 2010, 11:28 PM, said:

[size="2"]you said it's better to linearize the plasmid before transfection, If I understood properly, you mean cutting the plasmid with an enzyme, opening it then transfection.

Non-homologous end-joining will join many copies of linearized vector and then homologous recombination will integrate multiple copies of your vector (the drug selection gene and shRNA expression cassette) into host genome.  For example, a (unique) ScaI site in the Amp gene can be used to linearize the vector.  Also, you might need to do longer selection to ensure the recombination/integration.

Dear functional screens,

Thanks for ur useful comments. This time I will transfect the cells with linearized shRNAs. I will write here how my experiment went.

Now I have another question, for long time selection do u use the same concentration of antibiotic for whole time or u reduce the concentration after afew day?

Regards,
Banou

[Functional Screens]

grow cells under drug selection for a week, then it's up to you.  For example, you can have antibiotics all the time, or you can do one time selection every month to ensure all cells still have the constructs.

[banou]

Functional Screens, on Apr 26 2010, 05:01 PM, said:

grow cells under drug selection for a week, then it's up to you.  For example, you can have antibiotics all the time, or you can do one time selection every month to ensure all cells still have the constructs.

Hi
I got my stable transfected cells, The RT-PCR results show almost 80% knockdown for Dicer. It looks nice but I have encountered a strange finding that in Dicer knockdown cells my gene of interest is downregulated ( but we expected upregulation) the PCR has been done several times and in all of them the it shows down regulation..could any one find an explanation for this finding.
Thanks,
Banou