What should I do to clarify my sample? Probably my samples are contaminated by polysaccharides. I washed samples with 70% ethanol many times, I centrifuged samples and precipitated again by isopropanol and still white pellet appearing... The ratio 260/280 is 1.8, but ratio 260/230 is 0,5
You could try passing your RNA samples through a column and wash the polysaccharides out with 75% ethanol or the wash buffer provided in an RNA prep kit. I've never had this particular issue isolating RNA, but maybe investing in a RNA cleanup kit would save you time and money. What organism/ tissue are you isolating RNA from?
RNA precipitates in the presence of isopropanol, so the solution is expected to become cloudy. After the centrifugation you will always see a pellet (that is your RNA). I cannot see a problem here! 260/280 ration is perfect! The only problem is that your 260/230 ratio is low! It means that your sample is contaminated with organic solvents, like phenol (used on the first step of the extraction).
You can get low 260/230 ratio from polysaccharide contamination, not just phenol, Edmilson. I found this article to be helpful as this is often a problem for scientists trying to isolate RNA from homogenized plant tissue. Maybe it will help.
http://www.nanodrop.com/Library/T111-Detection-and-Avoidance-of-Polysaccharides-in-Plant-Nucleic-Acid-Extractions.pdf
Sean Seltzer I'm working on cucumber.
Today I checked my sample and RNA was degradated.
I know how isolated RNA looks like it is gel-like pellet. This pellet reminds me white, difficult to dissolve soap.
Thank you all!
sometimes the pellet can be white, this has never altered my 260/230 too much, but for plants too many polysaccharides can affect the 260/230 ratio. the protocol I linked to you was for russet potato and cucumber. Don't confuse low 260/230 with degraded RNA, it's completely unrelated and has to do with how pure your RNA is. The protocol I linked looks good for cucumber. I would try it with the high salt buffer they suggest to prevent the polysaccharides from precipitating with your RNA which can affect downstream experiments.
Dear All, I finally did it and my RNA looks good. Both ratios are OK. During the precipitation step I add sodium citrate and NaCl, and all the polysaccharides have disappeared.
Thank all of you for help.
Joanna Chojak-Koźniewska
I am facing the same problem with rice seed sample!
So could you tell me on isopropanol precipitation step, how much sodium citrate and NaCl you have added?
Gayathri Kks
During precipitation add to the aqueous phase 0.25 ml of isopropanol followed by 0.25 ml of a high salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl) per 1 ml of TRI RNA Extraction Reagent used for the homogenization. Mix the resulting solution, centrifuge and proceed with the isolation as described in the protocol. The modified precipitation effectively precipitates RNA while maintaining polysaccharides and proteoglycans in a soluble form.
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