glucose transporters

biology_06er · Post by **biology_06er** » Wed May 07, 2008 12:22 pm

glucose transporters

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Post by biology_06er » Wed May 07, 2008 12:22 pm

Hi there,

so I have to write up a report like thing on glucose transporters and how "to find expression of various glucose transporter isoforms in the cortex at the mRNA level"!!!...and we have to talk about what steps to take etcetc...umm have NOOO idea where to begin other than say use trizol to extract RNA from brain??...and isolate mRNA...buuut how to tell between the diff. isoforms...any help in terms of what i should be looking up online/articles/key words would be muuuch appreciated...

cheers
b_06er

MrMistery · Post by **MrMistery** » Wed May 07, 2008 3:19 pm

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Post by MrMistery » Wed May 07, 2008 3:19 pm

revers-transcribe the mRNA with polyT primers and then do real-time PCR with primers specific for each isoform gene...

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

mith · Post by **mith** » Wed May 07, 2008 6:32 pm

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Post by mith » Wed May 07, 2008 6:32 pm

If you want to skip the pcr part, use a microarray.

Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

MrMistery · Post by **MrMistery** » Wed May 07, 2008 8:43 pm

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Post by MrMistery » Wed May 07, 2008 8:43 pm

A microarray won't show how much it is expressed, only if it is present or not. It depends what you're looking for...

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

mith · Post by **mith** » Wed May 07, 2008 10:14 pm

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Post by mith » Wed May 07, 2008 10:14 pm

au contraire
From wikipedia excerpt
These molecules are used as probes to measure the relative abundance of the complementary sequences present in the samples (target) under study. This is done by hybridizing the two under high-stringency conditions and then detecting the sequences of the sample, which consist of a mix of either cRNA or cDNA commonly fluorescently labelled.

You can measure the intensity of the fluroescence with lasers.

Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

MrMistery · Post by **MrMistery** » Fri May 09, 2008 8:54 am

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Post by MrMistery » Fri May 09, 2008 8:54 am

I still don't see how. It either binds, or it doesn't.

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

canalon · Post by **canalon** » Fri May 09, 2008 12:40 pm

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Post by canalon » Fri May 09, 2008 12:40 pm

Microarrays are quantitative: Products are labeled during or after RT and since the intensity of the signal depends on the number of attached molecules. you can read deduce the relative expression of a gene compared to a standard (housekeeping gene usually) just as well as with qPCR.

Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

mith · Post by **mith** » Fri May 09, 2008 1:38 pm

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Post by mith » Fri May 09, 2008 1:38 pm

It's not simply one spot, each spot has a lot of attachment sites.

Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

MrMistery · Post by **MrMistery** » Fri May 09, 2008 2:34 pm

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Post by MrMistery » Fri May 09, 2008 2:34 pm

Uhum, got it. Thanks for the detailed info Patrick.
Sorry to bug you for nothing Dave

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

biology_06er · Post by **biology_06er** » Fri May 16, 2008 11:21 pm

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Post by biology_06er » Fri May 16, 2008 11:21 pm

hi, thx for the reply/s..so r those techniques the only ones possible? if u guys were gonna carry out this experiment is that wat you guys wld do? PCR and microarray?

mith · Post by **mith** » Fri May 16, 2008 11:45 pm

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Post by mith » Fri May 16, 2008 11:45 pm

mrna expression is standard for microarrays.

Living one day at a time;
Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr

MrMistery · Post by **MrMistery** » Sat May 17, 2008 10:12 am

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Post by MrMistery » Sat May 17, 2008 10:12 am

Cost would also be involved: where I work we don't afford microarrays(which was the main reason I didn't know exactly how a microarray works - I have never actually seen one). And I'm thinking the number of genes involved: if you want to study only 2 or 3 genes(as might be the case with your glucose transporters) Real time would work just fine. If you want more genes, using a DNA chip would save you a ton of time, since you can analyze a truck-load of genes at once.
But i'm really just guessing here. I'm not very familiar with either

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

glucose transporters

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