The TURBO DNA-Free Kit is Ambion’s answer to the dilemma of DNase inactivation without damage to RNA samples or affects on downstream reactions. The kit includes a powerful recombinant DNase, buffer and a proprietary inactivation reagent. The inactivation reagent is more convenient than performing a phenol extraction and eliminates the need for heating samples or adding a high concentration of EDTA. DNase buffer contains divalent cations that encourage degradation of the RNA when heated, making this a risky approach to removing DNase. Chelating the cations with EDTA is an option, but may cause problems in RT-PCR or other reactions that are concentration dependant. The inactivation reagent removes both the enzyme and the divalent cations, leaving the RNA suitable for other reactions. Phenol extraction followed by precipitation is another possibility, but the procedure is time-consuming and phenol is hazardous.
The three major advantages of TURBO DNA-Free are the reduced risk of sample degradation, the convenience and the high-affinity enzyme. After DNase treatment, the inactivation procedure involves a 2 min incubation and 1.5 min spin, followed by sample transfer to a new tube. The procedure can be completed in less than 10 min, depending on the number of samples. Another great feature is the potent TURBO DNase enzyme, engineered to have a greater DNA affinity. Because the TURBO DNase is a recombinant enzyme, RNase contamination is not a problem (although all Ambion products are carefully tested for RNase contamination). Some DNase enzymes are not RNase-free because wild-type enzymes are often isolated from pancreas, which is an RNase-rich tissue.
One potential disadvantage to this kit is the requirement for RNA transfer to another tube after inactivation to prevent PCR contamination. It is possible that some RNA may be lost in the process, which can be problematic for researchers isolating RNA from small samples (sample loss may occur with phenol extraction, as well). This kit is also a bit more expensive than traditional methods using standard DNase. For samples with minimal genomic DNA contamination, it may not be worthwhile to use the TURBO enzyme.
In my lab, we isolate RNA from human and rat brain tissue for quantitative RT-PCR, making effective DNase treatment critical. I have verified that RNA treated with these reagents is free of genomic DNA contamination by performing quantitative RT-PCR on RNA samples. As an extra precaution, I treat all of my samples as if they are highly contaminated, but with the new enhanced enzyme the standard treatment should be more than adequate.
Researchers who tried the DNA-free kit prior to 2004 may have been disappointed with the inactivation reagent, as I was. The instructions state that the aliquot of inactivation reagent should appear mostly white with minimal clear liquid. In practice, about 1 of every 10 aliquots was mostly white and I often worried that the enzyme was not inactivated. I had decided that I would use this product until I finished preparing the samples for the study that I was working on and then abandon it. I ordered more to finish the experiment and was initially skeptical when I saw the sticker indicating that it contained a new, improved inactivation reagent. Remarkably, the new version introduced last year is significantly better with no clear liquid. Therefore, I would encourage people who tried DNA-free in the past to consider using it again. The TURBO enzyme is available without the inactivation reagent and the inactivation reagent is also sold with Ambion’s standard DNase. However, the inactivation reagent is not sold separately.
Tara Lauriat
Graduate Student
Mount Sinai School of Medicine
New York, NY