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Why the fluoresence is dim or dispear in miRNA vector?


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#1 newmirnaman

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Posted 08 July 2009 - 06:45 AM

After the Red or Green fluorescence gene, I inserted my miRNA sequence flanked by 5' and 3'.
After I infected the cells, I found the controls are very bright fluoresence, but some of my miRNA vectors infected cells are dim or totally no fluorescence?
How to explain this?

Thanks in advance.

#2 Functional Screens

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Posted 08 July 2009 - 07:15 AM

This is the common problem if the miRNA precursor and marker gene both are on the single "EXON" transcript. The more miRNA processed, the more messenger RNA degraded, and you might see less marker gene expressed. The "natural" miRNAs are in the intron of coding sequences, or either intron or exon of non-coding sequences.

I have compared the miRNA expression system available in the market. The Open Biosystem's vector is everything (RFP, miRNA, IRES, and puro) on the same "exon"; the SBI's miRNA expression vector is two promoter driven:one for miRNA and one for marker gene (marker gene expression might not reflect the level of miRNA expression); The Biosettia's miRNA lentiviral expression vector is one promoter which expresses RFP::puro fusion and miRNA precursor is inserted in the Intron. The Invitrogen's pol II mir RNAi system does not count since it's used to express shRNA for target degradation, however, it's everything on the same "EXON". Hopefully this info helps.

Edited by Functional Screens, 08 July 2009 - 07:17 AM.


#3 newmirnaman

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Posted 08 July 2009 - 07:51 AM

Hi. Functional Screens

I used the lentivirus vector from openbiosystems. Plemir.

So, the fluoresence become dim means that the miRNA is expressing, right? Does this effect the miRNA to interact with its target genes?
Namely, this won't affect the results?

Because sometimes I can see my protein was down, sometimes not. This drove me crazy.
How can I make the conclusion?

BTW, which vector do you think is best?
Thanks. Functional Screens

Edited by newmirnaman, 08 July 2009 - 08:00 AM.


#4 Functional Screens

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Posted 08 July 2009 - 10:42 AM

Thinking of the positive side, the dimmer RFP expressed the better miRNA processed. The disadvantages are : 1. hard to track the transduction efficiency. 2. might not able to do selection especially the puro is expressed via IRES (gene expressed via IRES is ~10-fold lower than it was placed before IRES). However, all these should not affect the functional of miRNA, it works it works. Buit, you might want to generate higher titer virus to ensure the reproducibility.

Have you tried reporter assay, it might help you to draw the conclusion.

Which vector is the best? Well, I am biased, so I will leave it to you to decide.




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