Padeasy vector

Total DNA was isolated from finely minced tissues (∼20 mg of liver, pancreas, brain and ∼15 mg of spleen) by using DNeasy 96 Blood & Tissue Kit from Qiagen, USA following the manufacturer’s guideline. Quantitive real time PCR was performed to determine the copy number of adenoviral genomic DNA in liver, pancreas, brain and spleen by using iTaq™ Universal SYBR^® Green Supermix from Bio-Rad, USA. The following primers were used based on a previous study [30 (link)]. The sequences of the forward and reverse primers were 5’-CCACCGATAGCAGTACCCTT-3’ and 5’-GACCAGTTGCTACGGTCAAA-3’, respectively. The PCR cycle consists of one cycle of 95°C for 3 min and then 40 cycles of 95°C for 10 s, 55°C for 10 s and 72°C for 30 s and one cycle of 95°C for 10 s. Purified pAdEasy vector (Agilent Technologies) with known copy number (2.2X10⁶ to 2.2X10^-1 copy) was used to prepare the standard curve for the determination of Ad viral DNA copy number in the samples. Each sample and standard were run in triplicate. A Bio-Rad CFX connect PCR detection system was used for real time PCR and data was anlyzed by Bio-Rad CFX manager. A negative control using molecular biology grade water (ThermoFisher Scientific, USA) instead of DNA template was included and the quantification cycle (Cq) values less than the negative control were considered as negative.