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. 2014 May 7;9(5):e96490. doi: 10.1371/journal.pone.0096490

Figure 2. Real-time monitoring of RNase activity by fluorescence resonance energy transfer (FRET) via short double strand RNA degradation.

Figure 2

A) Schematic illustration of the FRET based RNase assay. The duplex RNA (13 bp) was dual-labeled with a fluorescein (FAM) as donor and tetramethylrhodamine (TAMRA) as acceptor on each end. B) Normalized fluorescence spectrum of duplex RNA (13 bp) incubated with (green line) or without (red line) RNase/serum at 25°C for 15 min. C) Duplex RNA degradation assay measurement in real-time. Duplex RNA was annealed and incubated in annealing buffer at 25°C. RNase or serum was added at the time indicated. Fluorescence of the donor (FAM) was recorded in real-time (solid dots). Samples at different time (from left to right: 0 s, 10 s, 60 s, 180 s, 480 s) were collected and run in a denatured PAGE gel (Inset). Fluorescence was scanned on Typhoon showing the remaining amount of full length dsRNA.