In Vitro Transcription and RNA Hybridization

EcoRI-linearized FHV_T_Rluc and FHV_T_minus and XhoI-linearized pEAV221Δ [34 (link)] plasmids were used as templates for in vitro transcription. Transcripts of FHV_T_Rluc and FHV_T_minus corresponding to capture probes for minus and plus strands, respectively, and a transcript containing nucleotides 1-2042 of equine arteritis virus (EAV) genome were prepared using mMESSAGE mMACHINE T7 Transcription Kit (Ambion) according to the manufacturer’s instructions. In vitro transcription reactions to prepare ³²P-labeled transcripts of FHV_T_Rluc and FHV_T_minus consisted of 750 ng of the respective linearized plasmid, transcription buffer (Promega), 800 U/mL T7 RNA polymerase (Promega), 1000 U/mL RiboLock (Thermo Fisher Scientific), 5 mM DTT, 1 mM ATP, UTP, GTP and CTP, and 0.133 μM α-³²P-CTP (20 μCi) (Perkin Elmer, Ayer Rajah, Singapore). The reactions were incubated for 2 h at 37 °C followed by RQ1 RNase-free DNase treatment (40 U/mL; Promega) for 35 min at 37 °C and inactivation by RQ1 DNase STOP solution (Promega) for 10 min at 65 °C. Unincorporated label was removed using RNase-free Micro Bio-Spin P-30 Gel Columns (Bio-Rad). Hybridization with the ³²P-labeled RNA transcripts and IVRA products was performed as described [34 (link)].