[biology_06er]

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]

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

[mith]

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

[MrMistery]

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

[mith]

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.

[MrMistery]

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

[canalon]

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.

[mith]

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

[MrMistery]

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

[biology_06er]

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]

mrna expression is standard for microarrays.

[MrMistery]

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

[canalon]

Mr Mistery is right:
-RTqPCR if you have a few genes. Depending on the machines you can multiplex 3 to 5 genes (and that would include a control housekeeping gene to normalize for variations in RT efficiency). The positive side is that you can work with a truckload of samples (say 40 in a 96 wells plates if you include duplicates, controls and normalization curve) everyday (a common program would run in 2 to 3 hours, so you can easilly run 2 to 3 plates a day)
-plenty of genes, then it becomes worthy to move to microarrays. But it is usually harder to work with a truckload of samples, since the experiment is time consuming and demands a lot of work. Someone I know was considering 6 samples a day a ggod result. But 100’s of gene per sample. There is a trade-off. Unless you can afford robots to do the work.

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