Protocol for recombinant rabies virus titration by quantitative PCR

Preparing high-titer virus and performing accurate titer determination are critical to subsequent experiments. However, not all applied recombinant rabies viruses, such as the L-deleted virus,¹ are equipped with fluorescent proteins for titration by fluorescence-activated cell sorting (FACS). Here, we present a quantitative reverse-transcription PCR (RT-qPCR) approach for titrating recombinant rabies virus. We describe steps for preparing standards for RT-qPCR, rabies virus genome RNA extraction, and reverse transcription of virus RNA. We then detail procedures for RT-qPCR for titration and stereotaxic rabies virus injection for titer verification.