Abi 7900 ht qpcr machine

The AAV titer was determined by quantitative PCR (qPCR) using the iTaq Fast SYBR Green supermix with ROX (Bio-Rad, Hercules, CA) in an ABI 7900 HT qPCR machine (Applied Biosystems, Foster City, CA, USA). A set of primers were designed to amplify a fragment in the CK8 promoter. The forward primer is 5′-AGCCCCTCCTGGCTAGTCAC-3′, and the reverse primer is 5′-CCTGAGTGTCTGTCTGTGCTGTG-3′. The qPCR reaction was carried out under the following conditions: 20 s at 95°C, followed by 40 cycles: 15 s at 95°C and 60 s at 60°C. A dissociation curve step was applied at the end of the reaction to confirm the primer efficiency using the following conditions: 15 s at 95°C, 15 s at 60°C, and 15 s at 95°C. The threshold cycle (Ct) value of each reaction was converted to the vector genome copy number by measuring against plasmid standard series representing 1 × 10⁶ to 1 × 10¹¹ vg/μL at a log₁₀ increments.

Genomic DNA was extracted from liquid nitrogen-frozen tissue samples. DNA concentration was quantified with a Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative TaqMan PCR assays were performed using the TaqMan Universal PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA) in an ABI 7900HT qPCR machine (Applied Biosystems, Foster City, CA, USA) to detect either the R1-R16 or R17-R23 junction in the vector genome (Figure S4). For the R1-R16 junction PCR reaction, the forward primer is 5′-TGGCCAGCATGGAAAAGCA-3′, the reverse primer is 5′-GGTGATCTCGGTCAGGTAGGT-3′, and the probe is 5′-CAACCTGCACAGCTACG-3′. For the R17-23 junction PCR reaction, the forward primer is 5′-TGCAAGCAGCTGTCCGA-3′, the reverse primer is 5′-AGATGCAGCCGCTTCCA-3′, and the probe is 5′-CTGGTCGCTCTGTTCTT-3′. The qPCR reaction was carried out under the following conditions: 10 min at 95°C, followed by 40 cycles: 15 s at 95°C and 1 min at 60°C. The Ct value of each reaction was converted to the vector genome copy number by measuring against the copy number standard curve of known amount of the pTR-CK8-hDys5 plasmid. The data were reported as the vector genome copy number per diploid genome.