RNase protection assay and agarose gel

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Ammie
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RNase protection assay and agarose gel

Post by Ammie » Thu Feb 14, 2008 2:30 pm

From Ambion technical bulletin #169: "Northern assays reqire the total RNA to be resolved on a denaturing agarose gel first, then transferred to a membrane and immobilized for subsequent hybridization. Nonisotopic RPAs which utilize probes labeled with modifyed nucleotides are transferred to a membrane from denaturing polyacrylamide gels for detection by a secondary detection scheme. Since different types of gels are used for these teqniques the mode of transfer is different in each case. Agarose gels are used for Northerns because of their wide range of resolving power and large loading capacity...."

I planned to make RPA but with an agarose formaldehyde gel, so I was a little bit worried when I read that. But there was not any explanation why PAGE gels is used for RPA and not agarose gel.

Anyone who knows?

And do you think it is ok if I continue with the plan to use agarose gel?

Thanks.

blcr11
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Re: RNase protection assay and agarose gel

Post by blcr11 » Thu Feb 14, 2008 3:17 pm

You can certainly transfer RNA to membranes from agarose-formaldehyde gels by capillary action. I don't know about electroblotting, at least I've never done it that way. I don't know if there is any reason why agarose might interfere with with RNAse protection or not; I can't think of any obvious reason why agarose gels can't be used for that purpose. Denaturing acrylamide gels, within their range, anyway, should give sharper bands and greater mechanical strength, and so are easier to use--plus you can transfer your nucleic acids to membranes with electroblotting, which is much faster than by capillary action.

Here is one protocol for transfer off agarose-formaldehyde gels:
http://www.protocol-online.org/prot/Pro ... l-781.html

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Post by Ammie » Mon Feb 18, 2008 3:11 pm

Thanks for your answer blcr11!

I have made a first try now, with a RNA transcript insted of total RNAs from cells as a pre-trial. And it works at least with agarose gel!

the PAGE will give a sharper band, but I think that it is sharp enough now. But how about the sensitivity? Could the detection limit be lower with PAGE?

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