8 replies to this topic

[Jean Lu]

hi!
My lab encounter wired problems in shRNA . we have several shRNAs that can knockdown their target genes at the mRNA level well. we measure them by real time PCR and has a 70-90% knock down efficiency. we design the primers of RT-PCR at the conserved region of the gene. However, the protein did not knockdown no matter how many days we tested . we examined at day3,day6,day9, and day 15 post infection. we are working on human embryonic stem cells renewal and mesenchymal stem cells differentiation. Could somebody give us  input? how to troubleshoot this? we appreciate your kind help and opinions!

Best Wishes,
Jean

[Ambinlab]

Dear All,

After spending more than 3 weeks, I got my cloning working. I got three plasmids with the right sequences which (should) express shRNA against my gene of interest.

I transfected 293s with my plasmids and was able to see the GFP, which was a marker on the vector. SO I am pretty sure that the cells were transfected efficiently.

I harvested them after 48h and 72h, but I could not see any knockdown effect on western blot.

Should I try RT PCR? Should I design more shRNAs against the same gene?

Any ideas would be highly appreciated

[pcrman]

Definitely, you should first detect mRNA level by RT-PCR, if no drop in mRNA, then the shRNA is not working, otherwise, probably the half life of your protein is very long.

[SJGradStudent]

Hi all,

I have been struggling for quite some time to see knockdown at the protein level. I have used both siRNA and shRNA (lentiparticles) and I can see a dramatic reduction (~80% K/D) in the target gene, but I can never detect the protein knockdown. The lenti system I use also has GFP included in the vector as a transduction control. I have tried multiple antibodies at different dilutions and have used a control/overexpressed samples to make sure I am looking at the correct band. I can see get overexpression, just not K/D.

Any ideas why this would be so?

Thanks,
Jordan

[scolix]

what is the turnover time for the protein of interest? Sometimes, you need to wait for sometime before you can see a reduction in protein levels.

[pcrman]

As Scolix said, the transfection duration could be critical for you to see drop in protein level. You can harvest cells between 3-5 days following transfection. Are there any isoforms of your gene? It could be that the siRNA is knocking down one of them, while your antibodies recognize other isoforms.

[zodiac1505]

We have had the same problem in our lab  with a couple of genes on which we used to work. One of the genes I worked on, I had to try out 9 different shRNAs before I found one which actually worked. And that took 2 years. We weren't able to pinpoint what the reason was but like pcrman said, my gene had 2 isoforms and so we felt that might have been one of the reasons.

[Nabin]

Ambinlab, on 17 January 2012 - 02:37 PM, said:

Dear All,

After spending more than 3 weeks, I got my cloning working. I got three plasmids with the right sequences which (should) express shRNA against my gene of interest.

I transfected 293s with my plasmids and was able to see the GFP, which was a marker on the vector. SO I am pretty sure that the cells were transfected efficiently.

I harvested them after 48h and 72h, but I could not see any knockdown effect on western blot.

If you have your vector with your GOI and GFp expressing sequence both of them together, what might have happened is, when your plasmid broke during insertion, it might have destroyed your GOI sequence. I had similar kind of problem with those vector, after transfection and keeping the cells in selection for more than two weeks, there were still plenty of cells in my 6 well plate, but the cells that were showing green in GFP were very few.. I continued my experiment and picked up the colonies (I was unsure which colonies to pick)  the non green colonies showed expresiion of my GOI but cells from green colonies were just like my regular cells. i used RT PCR for initial screening...

Nabin

Edited by Nabin, 24 August 2012 - 11:57 AM.

[mhj222]

We tend to think the relationship between mRNA levels and protein expression is fairly linear. But of course it isn't. There was a recent paper in Nature http://www.nature.co...ature11508.html which showed that protein levels can be increased with mRNA levels remaining constant due to the effect of an lncRNA which specifically improves the translation efficiency of a single mRNA. Turns out its also a great way to knock-up genes. see SINEUP