Figure 4

Similar copy numbers of ssAAV and scAAV in infected hearts. Serial sections of tissues used for microscopy were collected for real-time PCR analysis of AAV vector genomes. Genomic DNA (gDNA) was extracted from the tissues using a Qiagen DNeasy tissue kit according to the manufacturer's protocol. One microgram of extracted gDNA was used in all quantitative PCR reactions. The PCR conditions were 50 cycles of 94.8°C for 40 s, 37.8°C for 2 min, 55.8°C for 4 min, and 68.8°C for 30 sec. DNA samples were assayed in triplicate. The third replicate was spiked with CBA DNA at a ratio of 100 copies/μg of gDNA. If at least 40 copies of the spike-in DNA were detected, the DNA sample was considered acceptable for reporting vector DNA copies. Data were averaged by group and plotted with standard deviation for comparisons. No statistically significant differences were measured between any of the groups of scAAV and ssAAV treated animals.