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gyma

Member Since 16 Nov 2009
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Posts I've Made

In Topic: Lymphocyte culture trouble!!

Yesterday, 11:09 PM

make sure that you isolated the splenocytes correctly in the first place. 48 h of ex vivo culture wont cause death of all splenocytes generally, maybe just a portion. I suppose the problem is in your isolation procedures.

In Topic: Is overexpression a correct way to check shRNA knockdown efficiency?

04 January 2013 - 11:44 PM

bob1, on 28 December 2012 - 11:40 PM, said:

Yes, I was meaning checking the KD using endogenous levels in the 293s.

Just finished qPCR check and I was able to see the knockdown effect. However, none of the them had >50% knockdown of endogenous level, which is a bad news for me. I remembered that the vector that I used in the overexpression method (the successful one) is also CMV promoter. But in that case, I was able to see an almost 90% knockdown by western blot. I guess maybe the main reason is that the knockdown plasmid is just so effective that I can observe it by overexpression.

Shall I proceed with only less than 50% KD? Is there any methods to improve the KD? Can I expect some different result in cell lines like T-cells other than adherent cells?

In Topic: Is overexpression a correct way to check shRNA knockdown efficiency?

29 December 2012 - 12:23 AM

bob1, on 28 December 2012 - 11:40 PM, said:

Yes, I was meaning checking the KD using endogenous levels in the 293s.

thanks i will try that

In Topic: Is overexpression a correct way to check shRNA knockdown efficiency?

28 December 2012 - 08:12 PM

bob1, on 28 December 2012 - 06:39 PM, said:

Have you looked at the transcript and seen if there is KD?

No. Because previously I have used this method to check the knockdown effect and it worker well, I thought it could be a common way. Now I realize that previously I may have used a non-CMV promoter-based vector.
You mean if RNA level is detectable in eg 293 cells, I can still check it in 293?

In Topic: Is overexpression a correct way to check shRNA knockdown efficiency?

28 December 2012 - 04:31 AM

Thank you guys. But the problem is endogenous expression of these genes in 293 or other adherent cell lines are very low. Thats why I used overexpression. And these vectors dont have a fluorescent marker so I cant sort the positive cells out. Do you guys have any advice on this point? Thank you.

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