(NONE)

[Jagreet Kaur]

Hi
Thanks to every one for the advice and suggestions .Here are the replies I
had received.Sorry for the delay in posting them on to the net.
Date: Thu, 20 Jan 2000 09:58:23 -0500    From: Rajeev
Verma<rav94001 at uconnvm.uconn.edu>

I have tried the isolation of RNA from siliques,and it appears to work
okay, though have not yet run northerns with the preps. Here's what I
used;

Grind 2-4g fresh siliques in RNA free mortars&pestles.

Homogenise in 15ml extraction buffer
-50mM tris/HCL pH 8.0
-150mM NaCl
-5mM EDTA

Add 1.8ml of 20% SDS, invert to mix. Centrifuge at 10,000rpm in SS-34
rotor, 10mins at 4 degrees

Extract supernatant with 15ml buffered phenol, centrifuge at 5,000rpm,
SS-34, 5mins at 4 degrees

Extract the aqueous phase 2times with 15ml of a 1:1 mixture of buffered
phenol     and chloroform, centrifuge to partition.

Extract the aqueous phase with 15ml of chloroform, centrifuge to
partition.

Transfer aqueous to fresh tubes and add 0.3volumes of 10M LiCl,
precipitate overnight at 4 degrees.

Centrifuge at 18,000rpm, SS-34 for 15mins at 4 degrees.

Wash pellet (RNA) with 40ml cold 70% ethanol, centrifuge at 10,000rpm,
SS-34 for 20mins at 4degrees, decant ethanol and air dry the pellet.

Resuspend in appropriate volume (100-200uL) of sterile RNase free water.

Store preps at -80degrees.

**Note: if the preps appear colored or gummy, thisindicates precipitation
of pectic and other carbohydrate material in the prep. To remove these
just repeat the phenol, phenol/chloroform and choloform extraction and
precipitate with LiCl and ethanol wash the pellet.

Rajeev Verma
Wolf-dieter Reiter Lab
Molecular & Cell Biology
University of Connecticut
Storrs, CT 06269, USA
Email: rav94001 at uconnvm.uconn.edu






Our lab has used TRIZOL to extract RNA from all parts of Arabidopsis with
few
problems so it is possible.  I could suggest other  excellent procedures
with
high yields, but first I would need some answers to the following
questions:

How did you conclude that the problem is in isolating the RNA?

Did you also try to extract RNA from other tissues?

Jim Tokuhisa
Max Planck Institut fr Chemische kologie
Carl-Zeiss-Promenade 10
D-07745 Jena
Germany

Phone:  +49-3641-64-36-51
FAX:  +49-3641-64-36-50
E-mail:  tokuhisa at ice.mpg.de



Date: Thu, 20 Jan 2000 12:00:02 +0100
From: Rik van Gorsel <rvgorsel at mpc186.mpibpc.gwdg.de>
Subject: Re: RNA isolation from siliques

Dear Jagreet,

Very soon I will also start isolating RNA from siliques and I would be
interested to know at what stage of isolation you expect the problem to
be. I was planning to start with the TRIZOL protocol given on the AFGC
web site (http://afgc.stanford.edu/afgc-array-rna.html). Maybe you can
compare that with the version you are using (or maybe it is the same). I
will let you know my experiences after my first run. I am interested to
hear what suggestions you get from other newsgroup members.

best regards, Rik

--
Dr. Rik van Gorsel

Date: Thu, 20 Jan 2000 16:05:58 WES
From: Bart.Thomma at agr.kuleuven.ac.be
To: Sonti at ccmb.ap.nic.in
Subject: RNA from siliques
Dear Jagreet,

I read you have problems isolating RNA from arabidopsis siliques.
I have done this also a few years ago, and I never met any problems. I
used
this RNA for RT-PCR and some results are shown in:

Thomma, B.P.H.J., Broekaert W.F., 1998. Tissue-specific expression of
plant
defensin genes PDF2.1 and PDF2.2 in Arabidopsis thaliana. Plant Physiol.
Biochem. 36, 533-537.

Penninckx ,I.A.M.A., Eggermont, K., Terras, F.R.G., Thomma, B.P.H.J., De
Samblanx, G.W., Buchala, A., Mtraux, J.-P., Manners, J.M., Broekaert, W.F.
,
1996.  Pathogen-induced systemic activation of a plant defensin gene in
Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8,
2309-2323.

I think it is quite crucial to add phenol as quickly as possible to the
disrupted tissue while you are extracting, because seeds can contain high
RNase concentrations. Our laboratory published an article for RNA
extraction
Eggermont, K., Goderis, I.J., Broekaert, W.F. , 1996.  High throughput RNA
extraction from plant samples based on homogenisation by reciprocal
shaking in
presence of a sand/glass bead mixture. Plant Mol. Biol. Rep. 14, 273-279.

Best wishes and good luck,

Bart.

Bart Thomma
FA Janssens Laboratory of Genetics
K. Mercierlaan 92
3001 Heverlee-Leuven
Belgium (Europe)

Tel:  +32-16-32.96.58
Fax: +32.16+32.19.66





Date: Fri, 21 Jan 2000 08:40:14 +0900
From: Derek Bartlem <bartlem at abs.agr.hokudai.ac.jp>
To: sonti at ccmb.ap.nic.in
Dear Jagreet,

We have a protocol that works well for isolation of RNA from siliques
(whole siliques including seeds).  I guess this could also be used
successfully for just the silique tissue on its own.  The protocol can be
found at our lab home page, the address is:

http://arabi4.agr.hokudai.ac.jp/arabie/arabie.html

You will see at the top of the page in the new section, there is a note
that seed RNA extraction protocol has been added and you can follow this
link to see the protocol.  You can also get to this page by following the
link for General Molecular Protocols lower down the page.  This protocol
was developed for silique/seed RNA extraction as we found the methods we
normally use for extracting from other tissues were no good.

I hope this helps.  I am also interested to hear about other silique RNA
extraction protocols, would you be able to forward me any other useful
replies you may get?

All the best,
Derek

********************************************
Derek Bartlem
Laboratory of Molecular Biology
Faculty of Agriculture, Hokkaido University
Kita-9, Nishi-9, Sapporo 060-8589 JAPAN
Fax: +81-11-706-4932
e-mail: bartlem at abs.agr.hokudai.ac.jp
http://arabi4.agr.hokudai.ac.jp/arabie/arabie.html


---