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microRNAs and mRNAs. Interpreting gene expression profiles


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#1 netdavid

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Posted 24 February 2010 - 02:05 AM

Hi guys,
I'm a newbie and may be I'm doing a stupid question but I really need to understand. I will do an example to make me understand. Let say that in a gene expression profiling I see that a given mRNA is equally upregulated in a set of samples. At the same time I know that only in one of these samples are present the microRNAs (let say 4) that are potentially targeting it (targetscan prediction). May I say that likely in this sample I don't have the protein even though the mRNA level is high? Thank you in advance for your help....

#2 Fizban

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Posted 24 February 2010 - 07:42 AM

Hi guys,
I'm a newbie and may be I'm doing a stupid question but I really need to understand. I will do an example to make me understand. Let say that in a gene expression profiling I see that a given mRNA is equally upregulated in a set of samples. At the same time I know that only in one of these samples are present the microRNAs (let say 4) that are potentially targeting it (targetscan prediction). May I say that likely in this sample I don't have the protein even though the mRNA level is high? Thank you in advance for your help....


I wouldn't say "likely". Let's say remotely possible but yes, you could think that. But always beware of target prediction, it is VERY far from being reliable.
If there are not known miRNAs targeting your mRNA you have nothing else to do but try.
Fiz

#3 netdavid

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Posted 25 February 2010 - 01:56 AM

Hi guys,
I'm a newbie and may be I'm doing a stupid question but I really need to understand. I will do an example to make me understand. Let say that in a gene expression profiling I see that a given mRNA is equally upregulated in a set of samples. At the same time I know that only in one of these samples are present the microRNAs (let say 4) that are potentially targeting it (targetscan prediction). May I say that likely in this sample I don't have the protein even though the mRNA level is high? Thank you in advance for your help....


I wouldn't say "likely". Let's say remotely possible but yes, you could think that. But always beware of target prediction, it is VERY far from being reliable.
If there are not known miRNAs targeting your mRNA you have nothing else to do but try.
Fiz

Thank you Fiz for your reply........I just got some results.......only in one sample these microRNAs are present. So now I have high mRNA expression in all the sample but and high microRNA expression (that supposedly targeting it) only in one of them (which of course behave differently). Unfortunately no antibodies are available for this protein. What should I do in your opinion? Is an shRNA against this mRNA a resonable approach? Thank you again

#4 Fizban

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Posted 25 February 2010 - 03:27 AM

[/quote]
Thank you Fiz for your reply........I just got some results.......only in one sample these microRNAs are present. So now I have high mRNA expression in all the sample but and high microRNA expression (that supposedly targeting it) only in one of them (which of course behave differently). Unfortunately no antibodies are available for this protein. What should I do in your opinion? Is an shRNA against this mRNA a resonable approach? Thank you again
[/quote]
first of all you can try to validate the interaction of the miRNAs you identified as interesting with 3'UTR of the putatively regulated mRNAs. that's basic molecular biology, you just have to clone the whole (or just a part) UTR downstream of a reporter gene (there are reporter constructs commercially available) and test if your miRNA is able to directly downregulate gene expression.
The Ab is something you must have. maybe there are Abs not working for WB but working in IF....otherwise you can have them made but could be long and expensive: better be sure that you have a real mRNA/miRNA interaction before!
Mind well: the reporter assay WONT tell you that what you see is real in your cells/tissue of interest, just that it is possible in the real world.
Hope it helps
Fiz

#5 netdavid

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Posted 25 February 2010 - 08:29 AM

Thank you Fizban,
I will do it and I will let you know.......




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