Notes: In this study, an in vitro assay was performed to examine the ribonuclease activity of the herpes simplex virus virion host shutoff (vhs) protein. To test whether a purified GST-vhs fusion protein exhibited RNase activity, the entire 3’ UTR of human IEX-1 mRNA (a known target of vhs) was labeled with ³²P and then incubated at 30°C with either GST-vhs or GST alone. Samples were taken at various time points and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The GST-vhs fusion protein displayed RNase activity. When RNasin^® Plus RNase Inhibitor was added to the reaction, no degradation products were seen, confirming that the nuclease activity of GST-vhs could be blocked by an RNase inhibitor. (3391)

Notes: To prepare single rat NK cells for reverse transcription, the cells were sorted by flow cytometry and lysed using 0.5% NP-40, 2.2µl of 5X M-MLV Reverse Transcriptase Buffer, 0.3µl of 0.1mol/l DTT, 5.5µl DEPC-treated water and 1U of RNasin^® Plus RNase Inhibitor. After a 30-minute incubation on ice, the lysates were heated to 65°C for 90 seconds, cooled to 22°C for 3 minutes and placed back on ice. For the RT reaction, 1U RNasin^® Plus RNase Inhibitor, 60 U of M-MLV Reverse Transcriptase, 2.5mmol/l dNTPs and 100µM primers were added to these single-cell lysates. (3390)

Notes: RNasin^® Plus RNase Inhibitor was added to RNA-DNA ligation reactions and in vitro transcription reactions to protect RNA molecules in both reactions.  For in vitro transcription reactions, 1 unit of RNasin^® Plus RNase Inhibitor was added to the reaction buffer.   (3257)