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littlecell's Content

There have been 5 items by littlecell (Search limited from 25-September 19)


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#99252 microRNA knockdown its target by western blot but can't inhibit the 3'UT

Posted by littlecell on 01 February 2011 - 07:05 AM in siRNA, microRNA and RNAi

Who did you buy your 3'UTR luciferase vector from?

The 3'UTR luciferase vector is pMIRGLO from Promega. This vector contains both firefly and renilla luciferase.



#99171 microRNA knockdown its target by western blot but can't inhibit the 3'UT

Posted by littlecell on 31 January 2011 - 01:08 PM in siRNA, microRNA and RNAi

Hi everyone,
I overexpressed my microRNA in the tumor cell line and did see 50-80% knockdown of its target proteins by western blot. So just like every publication, I tried to confirm the specificity with 3'UTR luciferase assay. For this purpose, 293T cells were seeded in 6-well plates. Each well was co-transfected with 1 ug of microrna expression plasmid or empty control plamsid and 10ng of 3'UTR-luciferase plasmid, which also already has another internal version of luciferase as the internal control for the transfection efficiency. So my questions include:
1. when microrna was overexpressed by the plamid (either transiently or stable), how long does it take to mature? considering it has to be transcribed into precursor, then processed to mature microrna, by then 3'UTR luciferase has been overexpressed (or not) ? is it too late to inhibit luciferase activity if co-transfection was used? So just transfect 293T or other cells with microRNA Vector for 24h before adding 3'UTR luciferase construct.
2. Is commerical synthetic microRNA molecules good at this trick? Since it doesn't need to be transcribed from the plasmid but just to be processed by Dicer or whatever to mature microRNA. So that's why we see most of the publication use this kind of magic molecules to do the 3'UTR luciferase assay?
3.What's the appropriate ratio if I still want to do this 3'UTR luciferase inhibition with plasmid based microRNA expression vector?
4. How long should I make the PLB lysate if I use the co-transfection protocol for the 3'UTR luciferase assay?

I also noticed the bad reproducibility of microRNA in terms of knockdown its targets. Even missile can miss its target, right?


Appreciate any suggestions.



#87543 nonreproducible knockdown efficiency with Plemir-micorRNA from Open Biosystem

Posted by littlecell on 21 September 2010 - 01:09 PM in siRNA, microRNA and RNAi

Were the repeats done with same infected cells or cells infected at a different time? Are you sure the infection efficiency is the same and miRNA overexpression is the same?



The same cell lines were infected at a different time by the same virus, which was also made at different time. I am not sure the infection efficiency is the same but it's always at least over 95% since there's RFP within the construct.
Right now I suspect that the virus titer may affect the results since each microrna targets so many genes and the knockdown efficiency may vary each time based on the accessibility of mature microRNA, and even the process efficiency of mature microRNA from the precursor.
It's seems that the only choice is try to increase the virus titer though I don't know it's right or not.



#87205 Why my micorRNA Knockdown can't be repeated?

Posted by littlecell on 17 September 2010 - 08:30 AM in Molecular Biology

I have tried to overexpress the interested microRNAs using the plemir vector from Open Biosystem. I did see very good knockdown (at least 70%) of at least one target for each MicroRNA by western blot. But the problem is that the knockdown at the protein level is not reproducible at all even the same cell line was infected with the microRNA virus. I have been seeing this inconsistent western blot result for at least 2 different micrRNA constructs.
Does any of you has the same experience when you using Plemir from Open Biosystem?
Appreciate your suggestions and advice.



#86897 nonreproducible knockdown efficiency with Plemir-micorRNA from Open Biosystem

Posted by littlecell on 14 September 2010 - 01:38 PM in siRNA, microRNA and RNAi

I have tried to overexpress the interested microRNAs using the plemir vector from Open Biosystem. I did see very good knockdown (at least 70%) of at least one target for each MicroRNA by western blot. But the problem is that the knockdown at the protein level is not reproducible at all even the same cell line was infected with the microRNA virus. I have been seeing this inconsistent western blot result for at least 2 different micrRNA constructs.
Does any of you has the same experience when you using Plemir from Open Biosystem?
Appreciate your suggestions and advice.




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