Commonly used methods for removal or inactivation of DNase after digestion include: heat inactivation, proteinase K treatment followed by phenol:chloroform extraction, chelation of essential ions with EDTA, and purification using a glass-filter binding method such as RNAqueous (see the sidebar at right, "RNA Isolation for RT-PCR). Each of these inactivation or removal methods has its drawbacks.
Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75C. Although this method appears straightforward, the divalent cations in the DNase digestion buffer can cause (chemically-induced) strand scission of RNA when heated. Studies at Ambion have shown that much of an RNA sample is destroyed when heated to 80C for 5 minutes in the presence of 2.5 mM MgCl2 and 0.1 mM CaCl2 (salts typically found in DNase I digestion buffer).
Proteinase K treatment
and organic extraction: Proteinase K treatment followed by
phenol:chloroform extraction is probably the most rigorous method
for DNase inactivation and removal, but it is time-consuming, and
organic extractions often cause some sample loss. Sample loss can be
minimized by back extraction of the phenol:chloroform phase, but
this adds another step to an already time-consuming procedure.
Additionally, many people prefer to avoid working with hazardous