We have used the RNaseAlert procedure at Ambion to determine the extent of RNase contamination in two different preparations of DNase I. It is critical to have RNase-free preparations of DNase I when eliminating genomic DNA contamination from RNA preparations. Both Ambion's RNase-free DNase I (Catalog #2222) and DNase I from another supplier (Supplier S) were compared in a microplate assay using a SpectraMAX Gemini XS spectrofluorometer. First a positive control assay was set up in which 0.5, 5, and 50 pg of RNase A were used to digest the control substrate and create a standard curve (Figure 1). Experimental samples containing different amounts of DNase were then tested with RNaseAlert. Reactions were monitored in real-time at 37°C over 30 min in 5-minute increments. As shown in Figure 2, the RNase activity of Supplier S' DNase I (600 ng/reaction) is comparable to the positive control reaction (Figure 1) containing 50 pg of Ambion's RNase A. In contrast, Ambion's DNase I shows no elevation in signal fluorescence over background, even when 4.4 µg (800 U) of DNase I enzyme is used (Figure 3). This result supports Ambion's claims of supplying DNase I that is RNase-free even when the enzyme is used at 400X the normal use concentration.
Figure 1. RNaseAlert¹ Substrate Cleavage with RNase A
. Relative fluorescence units (RFU) generatd by digesting the RNaseAlert¹ substrate with 0.5 pg, 5 pg and 50 pg of RNase A. The substrate contains a fluorophore and a quencher and will fluoresce when cleaved by RNases.
Figure 2. RNaseAlert¹ Substrate Cleavage with Supplier 'S' DNase I
. Relative fluorescence units (RFU) generated during incubation of the RNaseAlert¹ substrate with DNase I from supplier 'S'.
Figure 3. RNaseAlert¹ Substrate Cleavage with Ambion DNase I
. Relative fluorescence units (RFU) generated during incubation of RNaseAlert¹ substrate with Ambion's DNase I. Lack of fluorescence indicates no RNase present in the sample.