MmuPV1 E6/E7 mRNA Detection by RNAscope

Custom RNA in situ hybridization probes were developed to detect the full-length E6/E7 mRNA sequence of MmuPV1 by Advanced Cell Diagnostics for use with their RNAscope assay kit. CISH was performed according to the manufacturer's instructions for RNAscope2.0 as previously described (37 (link)). Briefly, formalin-fixed, paraffin-embedded (FFPE) mouse tail tissue sections were pretreated with heat and protease prior to hybridization with the probe. If treated with RNase A (Qiagen), slides were incubated for 30 min at room temperature with RNase A in PBS (10 mg/ml). Slides were subsequently washed three times in diethyl pyrocarbonate (DEPC)-treated water for 5 min per wash. To ensure RNA integrity and assay procedures, adjacent sections were also hybridized with a probe for the endogenous housekeeping gene ubiquitin and the bacterial gene dapB (negative control). After washing, a horseradish peroxidase (HRP)-based amplification system was then used to detect the target probes, followed by color development with 3,3′-diaminobenzidine (DAB).