DNA-free™ Kit From Applied Biosystems

RNA-based analysis methods such as RT-PCR, microarray analysis and Northerns are now a mainstay application in many labs. In order to achieve both accurate and reliable results, it is of the utmost importance to remove all trace levels of contaminating DNA from samples of RNA. No method of RNA isolation can extract RNA that is completely free from contaminating DNA and RNA isolated from tissues such as spleen or thymus may contain relatively high levels of DNA. The DNA-free™ Kit from Applied Biosystems has been specifically designed to remove contaminating DNA and subsequently DNase and divalent cations from an RNA sample, leaving any contaminated DNA digested to levels below the limit of detection by routine PCR.

The kit contains a recombinant DNase I (rDNase I), an optimized DNase reaction buffer, DNase Inactivation Reagent and Nucelase-free water. All the components are ready to use, following thawing and brief mixing. Unlike some other methods for removal of DNA contamination, in DNA-free™, the DNase is removed in a manner that does not require phenol/chloroform extraction, alcohol precipitation, heating or the addition of EDTA, meaning samples can be effectively treated with minimal disruption. The kit is capable of removing trace to moderate amounts of contaminating DNA (up to 50 ug DNA/mL RNA) and the process is relatively quick and easy.

Depending on the amount of contaminating DNA and nucleic acid concentration of the sample, there are two DNase digestion conditions. In all cases however, the reaction size can be between 10 and 100 ul and the reaction can take place in either 0.5 ml reaction tubes or 96-well plates, for convenience. Briefly, the protocol involves addition of DNase digestion reagents, a 37°C incubation for 20-30 minutes, followed by addition of re-suspended DNase inactivation reagent. The sample is then incubated at room temperature for 2 minutes, centrifuged at maximum speed and the RNA transferred to a fresh tube. In my experience the whole process can take as little as 35-40 minutes per sample.

I have used the kit to treat a number of RNA samples that were positive for trace amounts of contaminating DNA. The protocol was quick and easy to follow and required minimal hands on time. In the protocol a reaction volume between 10 and 100 ul is recommended. I used reaction sizes of both 20 ul and 50 ul, both of which were volumes that proved easy to work with. The main disadvantage of the kit is in the last stages of the protocol, where the sample is centrifuged to pellet the DNase Inactivation Reagent and the RNA containing supernatant must be removed without disturbing the DNase Inactivation Reagent. This step results in some loss of sample and therefore I would recommend using at least a 20 ul reaction volume. A reaction volume of 50 ul or more will minimize sample loss as a result of this step. After treatment, contaminating DNA was no longer a problem in my RT-PCR and NanoDrop readings for my RNA samples remained optimal.