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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Quantitation of Rabies Virus in Various Bovine Brain Structures
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Protocol for recombinant rabies virus titration by quantitative PCR.

He Zhang1, Xueping Gao1, Xiangyu Ge1

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Accurately titrating recombinant rabies virus is essential for experiments. This study introduces a quantitative reverse-transcription PCR (RT-qPCR) method for precise rabies virus titration, even without fluorescent markers.

Keywords:
Biotechnology and bioengineeringMolecular BiologyNeuroscience

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Area of Science:

  • Neuroscience
  • Virology
  • Molecular Biology

Background:

  • Accurate high-titer virus preparation and titration are crucial for reproducible experimental outcomes.
  • Standard titration methods like fluorescence-activated cell sorting (FACS) are not applicable to all recombinant rabies viruses, particularly those lacking fluorescent proteins (e.g., L-deleted rabies virus).
  • This limitation necessitates alternative, reliable methods for quantifying viral loads.

Purpose of the Study:

  • To develop and validate a quantitative reverse-transcription PCR (RT-qPCR) method for accurate titration of recombinant rabies viruses.
  • To provide a detailed protocol for implementing this RT-qPCR approach, including standard preparation and RNA extraction.
  • To demonstrate the utility of RT-qPCR for rabies virus titration in the context of neuroscience research.

Main Methods:

  • Development of a quantitative reverse-transcription PCR (RT-qPCR) assay.
  • Preparation of standard curves using known concentrations of rabies virus RNA.
  • Isolation of rabies virus genome RNA from viral preparations.
  • Reverse transcription of viral RNA into complementary DNA (cDNA).
  • Real-time PCR amplification and quantification of viral cDNA.
  • Verification of titers using stereotaxic rabies virus injection in animal models.

Main Results:

  • The developed RT-qPCR method provides accurate and reliable quantification of recombinant rabies virus titers.
  • The protocol details essential steps for standard preparation, RNA extraction, and reverse transcription, ensuring reproducibility.
  • Stereotaxic injection experiments confirmed the titer verification achieved through the RT-qPCR method.

Conclusions:

  • Quantitative RT-qPCR offers a robust and widely applicable alternative for titrating recombinant rabies viruses, especially those lacking fluorescent reporters.
  • This method enhances experimental accuracy and reproducibility in neuroscience and virology research involving rabies virus.
  • The detailed protocol facilitates the adoption of RT-qPCR for routine viral titration in research laboratories.