Problems with RNA extraction from Sorghum anthers/pollenRNA pollen sorghum
Posted 15 November 2012 - 05:21 PM
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!
Posted 16 November 2012 - 07:03 AM
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:
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:
A longer, more involved protocol:
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.