Protocol for recombinant rabies virus titration by quantitative PCR

He Zhang; Xueping Gao; Xiangyu Ge; Xiao Wang; Minghui Song; Xia Zhang; Lei Jin

doi:10.1016/j.xpro.2024.103567

Protocol for recombinant rabies virus titration by quantitative PCR

STAR Protoc. 2025 Jan 22;6(1):103567. doi: 10.1016/j.xpro.2024.103567. Online ahead of print.

Authors

He Zhang¹, Xueping Gao², Xiangyu Ge², Xiao Wang², Minghui Song³, Xia Zhang², Lei Jin⁴

Affiliations

¹ Lingang Laboratory, Shanghai 200031, China. Electronic address: zhanghe@lglab.ac.cn.
² Lingang Laboratory, Shanghai 200031, China.
³ Lingang Laboratory, Shanghai 200031, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
⁴ Lingang Laboratory, Shanghai 200031, China. Electronic address: jinlei@lglab.ac.cn.

PMID: 39847487
DOI: 10.1016/j.xpro.2024.103567

Abstract

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.

Keywords: Biotechnology and bioengineering; Molecular Biology; Neuroscience.