RNA extraction using trizol

Principle reagents:
guanidinium isothiocyanate (powerful protein denaturant) -> inactivation of RNases
acidic phenol/chloroform(toxic) -> partitioning of RNA into aqueous supernatant for separation

Note: low pH is crucial since at neutral pH DNA not RNA partitions into the aqueous phase.


Nucleic acid (RNA, DNA) partitions in the aqueous phase, while protein partitions in organic phase.

For RNA purification, the pH is kept at around pH 4, which retains RNA in the aqueous phase preferentially. For DNA purification, the pH is usually near 7, at which point all nucleic acids are found in the aqueous phase.


Acid phenol specifically leaves RNA in the aqueous phase.  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.  In this case, DNA dissolves in the organic phase (like dissolves like).  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.

http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/PHENOL-CHLOROFORM.pdf