Purification of RNA Using TRIzol (TRI Reagent)

TLDR

TRIzol is a recent, monophasic phenol/guanidinium reagent that deproteinizes RNA and, because it solubilizes material and denatures proteins, is especially useful when cells or tissues contain high RNase levels or when cytoplasmic and nuclear RNA separation is impractical, allowing simultaneous purification of RNA, DNA, and protein from a single sample. After solubilization, adding chloroform induces phase separation, extracting protein into the organic phase, DNA to the interface, and leaving RNA in the aqueous phase. TRIzol effectively isolates small RNAs such as microRNAs, piwi-associated RNAs, and endogenous siRNAs, but its expense and the difficulty of resuspending RNA pellets make it less suitable when regular phenol extraction is feasible.

Abstract

TRIzol solubilization and extraction is a relatively recently developed general method for deproteinizing RNA. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. TRIzol (or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name TRIzol). TRIzol extraction is also an effective method for isolating small RNAs, such as microRNAs, piwi-associated RNAs, or endogeneous, small interfering RNAs. However, TRIzol is expensive and RNA pellets can be difficult to resuspend. Thus, the use of TRIzol is not recommend when regular phenol extraction is practical.