9 replies to this topic

[gyma]

Hi I have a strange problem here. I got two shRNA sets targeting two different genes, each with 5 shRNAs. After I confirmed the sequence, I overexpressed 0.5 ug of vectors that express these two genes and 1 ug of each shRNA in 293FT. Unexpectedly, none of the 10 shRNA worked. I didn't see even a slight knockdown by western blot. This is so wierd because some of these shRNAs have been validated by the company. Am I using the wrong way or is it simply that all 10 shRNAs were ineffective? Thank you in advance.
happy holidays.

[pcrman]

I don't think you can overexpress a gene and check its knockdown efficiency by its shRNAs because the exogenous expressed genes  can give you very high level of expression which may mask any effect exerted by the shRNAs. You can just check endogenous gene expression without using an overexpressing vector.

[bob1]

I agree, using overexpression vectors will not show you the result you want, especially if the vectors have viral promoters.  At best you would need to use vectors with native human/mouse or similar promoters.

[gyma]

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.

[bob1]

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

[gyma]

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?

[bob1]

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

[gyma]

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

[gyma]

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?

[bob1]

The only ways to improve would be to try different amounts of siRNA and/or some different siRNAs against the same gene(s).