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Array quality RNA from Arabidopsis siliques

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Tue May 4 15:26:46 EST 2004


<x-flowed>Gill,

I have had very good luck with an RNA protocol from Chen et al (1993)
Plant Molecular Biology Reporter 11:113.  The protocol calls for 1-3
g of tissue, but I have scaled down pretty successfully for 250-500
mg tissue in 5 ml of extraction buffer.

Although we haven't used it for arabidopsis siliques I have used it
for developing seed pods from alfalfa and we have used it for a
number of tissues that didn't work at all well with RNeasy Plant or
Triazole protocols.  It's a pretty easy prep and we pretty much use
it as our standard large scale prep.

The RNA seems pretty clean based on 260/280 ratios, and has worked
well both for blots and for making cDNA.  I can't say if it might
work well for arrays, but it might be worth a shot.  This may at
least give you a prep that could be further cleaned up with a column
based prep to give the quality you need.

Hope this helps.

Mike

>Hi everyone,
>
>I am currently trying to obtain RNA from developing Arabidopsis
>siliques for use in microarray. However, it seems that once the seeds
>in the siliques have completed mucilage production it is very hard to
>obtain RNA which can be labelled successfully. Mucilage consists of
>large amounts of pectin and I am assuming that this polysaccharide is
>what is causing the problem.
>
>If anyone has a protocol which they have used for tissues with high
>polysaccharide, which gives RNA of array quality then I would be very
>grateful if you were willing to share!
>
>Cheers,
>
>Gill
>
>Dr Gillian Dean
>Post Doctoral Fellow
>Dept of Botany
>6270 University Boulevard
>Vancouver
>V6T 1Z4
>Canada
>Tel: (001) 604-822-2437
>FAX: (001) 604 822-6089


-- 

Michael L. Sullivan, Ph.D
Research Plant Molecular
Geneticist
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
---
</x-flowed>



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