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Problems with RNA extraction from Sorghum anthers/pollen

RNA pollen sorghum

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#1 ricky56789

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Posted 15 November 2012 - 05:21 PM

Hey guys,
I'm having some problems with isolating RNA from anthers of Sorghum (Sorghum bicolor). I have tried a few protocols and no luck.

With the plant RNA reagent from invitrogen, a yellow/brown substance is in solution with the RNA (at the top of the solution after chloroform separation). It then precipitates with the RNA when isopropanol is added. I end up getting little to no RNA.

I have tried a protocol for high starch and the qiagen plant columns with no luck either. I dont know what is contaminating the aqueous phase. Any suggestions would be great!

Thanks
Ricky

#2 David C H

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Posted 16 November 2012 - 07:03 AM

I assume some class of polysaccharide is causing the problem; plants make such a variety of compounds that it is difficult to say. CTAB is commonly used to remove polysaccharides during isolation procedures.

First, with plant material, less is more. Use less tissue that what is recommended; if the protocol asks for 100mg of tissue, use 30-60mg instead.

Download the supplemental material at this link for a simple protocol that may help:

http://www.springerl...8387340u468h74/

The columns from the Qiagen kit (or other silica columns suitable for binding RNA) can be used with this protocol.

Here is a similar protocol that does not use columns:
http://www.sciencedi...166093408003273

A longer, more involved protocol:
http://www.springerl...33732khqg0736h/

It is probably best to avoid guanidine lysis buffers; most commercial kits, such as Qiagen, use a guanidine lysis buffer. These are very effective at neutralizing RNases, but have the tendency to co-precipitate some polysaccharides with RNA, preventing the RNA from entering the column; they may also cause some polysaccharides or other compounds to form a complex with RNA that prevents it from binding to the column. I have heard that some magnetic bead methods using guanidine (MagMAX or Agencourt SPRI) have less of a polysaccharide problem but I have not had the chance to test these systems.

Edited by David C H, 16 November 2012 - 07:07 AM.





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