We are trying to isolate RNA from frozen samples in alginate scaffolds with Trizol. We tried to increase the pH to speed up the dissolution of alginate. At pH 9, the most RNA was isolated, but the A260/A280 ratio was around 1.5. Is such a method with TRIZOL appropriate? Thank you.
Hello Maja Petre
Following the above method with TRIzol would not be appropriate. You never isolate RNA with increased pH. The purpose of acid phenol extraction is to selectively retain RNA in the aqueous phase while DNA partitions to the organic phase or coalesces at the interphase.
Please note that phase extraction of DNA and RNA is pH dependent. When the pH is greater than 7.0, both RNA and DNA will resolve in the aqueous phase. With a pH of less than 7.0, DNA more readily denatures and precipitates into the organic phase and phase interface, with RNA remaining in the aqueous phase. This is because as the pH decreases, the concentration of protons increases. DNA carries a negative charge because of the phosphate groups in its sugar-phosphate backbone, which are neutralized in acid by protonation. DNA will dissolve in the organic phase.
RNA, on the other hand, is not neutralized in acid because even though it also has a negative charge, it has exposed nitrogenous bases (it is single-stranded), which can form hydrogen bonds with water, keeping it in the aqueous phase. The low pH (acidic) of TRIzol controls to separate RNA from DNA and protein, while a high pH can cause RNA and DNA to be isolated together.
The 260/280 ratio of approx. 2.0 is generally accepted for RNA as pure. If the ratio is appreciably lower (A260/A280 ratio being 1.5), it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
Best.
Thank you. Based on the answer, I suppose it also doesn't make sense to put the solution after triazole on the RNeasy kit to isolate only RNA?
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