Starch Contamination during RNA isolation from wheat seeds - (Aug/18/2015 )
I am trying to isolate RNA from wheat seeds. We are using standard Trizol isolation method protocol but each time we try to isolate RNA we get very strong polysaccharides contamination. How can we get rid of polysaccharide contamination? We tried adding an additional step of wash PCI but it didn't help either. Please suggest, what can we try to eliminate this problem.
RNA isolation from plant is very tricky. Even we faced the same problem, which is still there though . But while searching for this problem, I came across this article. Just check if it is helpful in any way for your work-
Using less tissue may help, half as much as the protocol recommends. The yield will be lower, of course.
Trizol and many commercial kits include guanidine in their isolation buffers. The guanidine solutions are very effective at denaturing RNAses and their strong chaotropic nature results in coprecipitation of polysaccharides with nucleic acids.
CTAB-based methods can remove some polysaccharides during organic extraction. Precipitating nucleic acids in a potassium acetate buffer can selectively keep some polysaccharides in solution during the alcohol precipitation of nucleic acids. Care must be taken with CTAB buffers because these generally are not as good at inhibiting RNAses. However, when done properly, these methods can yield high quality RNA, suitable for RNAseq and real-time PCR.
Here is a method using CTAB and potassium acetate that yields high quality RNA and has been shown to work in many different species, Note that this also yeilds DNA and that will have to be removed if it is unwanted.
The following method uses different chemistry. I have not tried it (it was too time consuming for our purposes) but it may be suitable for your needs: