Nov 02, 2021

Public workspaceQuantitative PCR of Viral Abundance

DOI
dx.doi.org/10.17504/protocols.io.bzp4p5qw
  • 1California Institute of Technology
  • Mazmanian Lab
Jessica Griffiths
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DOI: dx.doi.org/10.17504/protocols.io.bzp4p5qw
Protocol Citation: Bryan Yoo, Jessica Griffiths, Sarkis Mazmanian 2021. Quantitative PCR of Viral Abundance. protocols.io https://dx.doi.org/10.17504/protocols.io.bzp4p5qw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 02, 2021
Last Modified: November 02, 2021
Protocol Integer ID: 54748
Funders Acknowledgements:
Department of Defense
Grant ID: PD160030
National Institutes of Health
Grant ID: GM007616 and DK078938
Aligning Science Across Parkinson’s
Grant ID: ASAP-000375
Center for Environmental and Microbial Interactions (CEMI)
Grant ID: n/a
Emerald Foundation
Grant ID: n/a
Heritage Medical Research Institute
Grant ID: n/a
Abstract
Protocol for quantifying viral abundance by PCR used in Yoo et al 2021
AAV-PHP.S:hSYN1-mNeonGreen was delivered systemically to wildtype mice at 6-8 weeks of age.
3-4 weeks following viral infection, 1cm of proximal, medial, and distal small intestine (SI) and colon were harvested and flash frozen in TRIzol (ThermoFisher Scientific, Waltham, MA-Cat. No. 15596018) and RNA was extracted per manufacturer’s instructions.
Viral ssDNA is present in the RNA fraction. Contaminating RNA was eliminated with RNase treatment (ThermoFisher Scientific, Waltham, MA-Cat. No. AM228) per manufacturer’s instructions.
Quantitative PCR was performed on viral DNA with primers against WPRE (Forward: 5’- GGCTGTTGGGCACTGACAAT-3’; Reverse: 5’-CCGAAGGGACGTAGCAGAAG-3’) and values were normalized to ubiquitous mitochondrial gene, mtRNR2 (Forward: 5’- CCGCAAGGGAAAGATGAAAA-3’; Reverse: 5’-TCGTTTGGTTTCGGGGTTTC-3’).
Plotted relative abundances are mean values across three regions sampled in either SI or colon.