Omnidoc imager

A DNA fragment corresponding to the RaTV 3′UTR under the T7 promoter was synthesized and cloned into the pIDT vector (IDT). Plasmids containing PCV 3′UTR and GFP gene fragments have been described previously [38 (link)]. For in vitro transcription, plasmids were first linearized by restriction digest and purified using a Monarch PCR and DNA Clean-up Kit (NEB). 3′UTRs were in vitro transcribed from 500 ng of plasmid DNA using a MEGAscript T7 Transcription Kit (Invitrogen). RNA was purified by LiCl precipitation and examined by electrophoresis in a 1 % denaturing agarose gel. RNA was then refolded in NEB3 buffer (85 °C for 5 min) and gradually cooled to 28 °C. For Xrn1 treatment, refolded RNA (1 µg) was incubated with 1 U Xrn1 (NEB) and 10 U RppH (NEB) in 20 µl of reaction mixture containing 1× NEB3 buffer (NEB) and 1 U µl^–1 RNasin Plus RNase Inhibitor (Promega) for 2 h at 28 °C. The reaction was stopped by adding 20 µl of Loading Buffer II (Ambion), heating for 5 min at 85 °C and placing on ice. The denatured RNA samples were loaded into 6 % polyacrylamide TBE-Urea gels (Invitrogen), and electrophoresis was performed for 90 min in 1× TBE. The gels were stained with ethidium bromide and imaged using an OmniDoc imager (Cleaver Scientific).

Plasmids were linearised by restriction digest and purified using Monarch PCR and DNA Clean-up Kit (NEB, USA). 3’UTRs were in vitro transcribed from 1 μg of linearised plasmids using MEGAscript T7 Transcription Kit (Invitrogen, USA) according to the manufacturer’s recommendations. RNA was purified by LiCl precipitation and analysed by electrophoresis in a 1.2% denaturing agarose gel. RNA was then refolded in NEB3 buffer by heating at 85 °C for 5 min followed by gradual cooling to 28 °C. The refolded RNA (1 μg) was incubated with 1U XRN1 (NEB, USA) and 10U RppH (NEB, USA) in 20 μL of reaction mixture containing 1x NEB3 buffer (NEB, USA) and 1 u/μL RNasin RNase Inhibitor (Promega, USA). Incubation was performed for 2 h at 28 °C. The reaction was stopped by adding 20 μL of Loading Buffer II (Ambion, USA), heating for 5 min at 85 °C and placing on ice. The entire volume was then loaded into 6% polyacrylamide TBE-Urea gels (Invitrogen, USA), and electrophoresis was performed for 90 min in 1xTBE. Gels were stained with ethidium bromide and documented using an Omnidoc imager (Cleaver Scientific, UK).