Detection of Viral Gene Expression

Gel-purified (gel extraction kit, Qiagen, Germantown, MD) chiA and v-cath PCR amplicons from agarose served as templates in PCRs incorporating only one (nested) primer for amplification of DIG-labelled ssDNA, complementary to either chiA or v-cath RNA, as described earlier [19 (link)]. RNA blots for each virus construct were pre-treated (30 min, 42 °C, 20 mL DIG Easy Hyb) and hybridized in 10 mL hybridization solution (15 h, 42 °C). Probes were labeled using the DIG nucleic acid detection kit and protocol (Roche, Little Falls, NJ) and 1 mL CSPD substrate (Roche) per blot. Chemiluminescence was detected with X-ray film (Kodak X-Omat).