Quantstudio absolute q

Mouse brain tissues were harvested in TRIzol (Invitrogen, USA), and total RNA was isolated as per the manufacturer’s recommendations. ZIKV genomes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript were quantified by digital PCR using a QuantStudio Absolute Q and a Combinati SARS-CoV-2 wastewater surveillance assay kit (Thermo Fisher Scientific). The probes for ZIKV and mouse GAPDH were labeled by 6-carboxyfluorescein (FAM) and VIC, respectively, which enables quantitation of both genes from the same RNA samples. The sequences of the primers and probe for reverse transcription and droplet PCR were previously published (40 (link)). The RNA samples were diluted 400-fold with water. Two microliters of the diluted samples was mixed with Combinati 1-step reverse transcription master mix and the primers and probes for ZIKV and mouse GAPDH, followed by reverse transcription, digital PCR, and data analysis using QuantStudio Absolute Q. RNA levels were expressed as ZIKV genome copies per one copy of GAPDH in the mouse brain tissue.

AAV viral copy number was determined by digital PCR of purified genomic DNA using custom-designed primers and TaqMan probes (Integrated DNA Technologies) (Table S1) on a QuantStudio Absolute Q digital PCR system (Thermo Fisher Scientific). The primers and probes anneal to the N-terminal and C-terminal Cas9 genes. A copy number reference assay for Tfrc (Thermo Fisher Scientific) was used to normalize genome copy number.

Mouse brain tissues were harvested in TRIzol (Invitrogen, USA), and total RNA was isolated as per the manufacturer’s recommendations. ZIKV genomes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript were quantified by digital PCR using a QuantStudio Absolute Q and a Combinati SARS-CoV-2 wastewater surveillance assay kit (Thermo Fisher Scientific). The probes for ZIKV and mouse GAPDH were labeled by 6-carboxyfluorescein (FAM) and VIC, respectively, which enables quantitation of both genes from the same RNA samples. The sequences of the primers and probe for reverse transcription and droplet PCR were previously published (40 (link)). The RNA samples were diluted 400-fold with water. Two microliters of the diluted samples was mixed with Combinati 1-step reverse transcription master mix and the primers and probes for ZIKV and mouse GAPDH, followed by reverse transcription, digital PCR, and data analysis using QuantStudio Absolute Q. RNA levels were expressed as ZIKV genome copies per one copy of GAPDH in the mouse brain tissue.