By Gary Latham and Meenal Patel, R&D Scientists at Ambion

Recently, Ambion introduced the RNase Alert¹ Kit. This simple, single-step assay tests solutions for contamination by a wide variety of RNases in only 30 minutes. RNase activity is visualized on a transilluminator or fluorometer.
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.