Quantitative PCR of Viral Abundance
Bryan Yoo, Jessica Griffiths, Sarkis Mazmanian
Abstract
Protocol for quantifying viral abundance by PCR used in Yoo et al 2021
Steps
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.
