Hi RNA experts,
I am trying to extract RNA from my blueberry fruit samples. They have been stored in -20 C for one or two months and then they were moved to -80 C. I used the Qiagen RNeasy Plant Mini Kit for extraction. The same lab/method/environment used for RNA extraction in other plant materials was used but I failed to extract RNA for these blueberry samples.
From QC, I got:
1. "too low" or close to 10.0 results (very low) from Qubit BR assays.
2. no visible bands from agarose gel electrophoresis.
3. 0.7-1.9 260/280 ratios from nanodrop (extensive variation).
What do you think the problem could be?
Could the high water content of the fruit be causing poor extraction?
Could storage temperature be the problem? Is it okay to store the samples before RNA extraction at -20 C?
Could the polyphenolics and polysaccharides be hindering extraction?
One can only speculate here what might be the problem. Looks like you might be right that polyphonics might have some influence, but kits generally have a lot of washing steps and contaminants are removed.
Have you tried to use fresh samples? Have you/anyone ever tried to extract RNA with your sample type? Fresh or freezed?
I would try to so that if things are not going well. Further, RNA extraction is always the best with fresh samples. With freezing or increase in the time duration between sample collection and extraction will just increase the chances of failure.
In my view, if your berry is still there, you could just repeat the extraction again. Few repetition only makes your problem vague. Besides, I suggest that next time you decide to extract RNA, use liquid nitrogen for instant cold, and store them in -80. And if you can use Grinding Mill triturate them and add TRIzol or TransZol and mix them and then store them or extract them, that will be better.
Our group also extracts RNA from fruit (in our case strawberry), and it seems like we have similar problem.
In my experience, samples should be stored in -80, and usually it's better to extract RNA ASAP. Unlike leaf sample or low-sugar fruits, berries seems fragile to store long time. But, it's just my opinion.
Also, as you wrote, fruits have high polysaccharides which need additional step. We used GeneAll Hybrid-R kit, and it recommends to use only supernatant of homogenized sample to mix chloroform.
Good luck for your experiments!
Few suggestion are here,
1. Collection of samples, has to be keep at -80C, that would be proper way you to get quality RNA.
2. The fruits may have high polyphenols, highly recommended to do with CTAB buffer to get good amount of RNA.
All the best
We usually isolate RNA from strawberry fruits. It gives a low amount by kits, so the Saltzman method is used (chloroform method). See below:
Article Expression patterns of ethylene and polyamine biosynthetic g...
Thank you everyone for your help. I used the Sigma Aldrich Spectrum RNA extraction kit and I got perfectly good extractions. I used the exact same samples.
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