Taqman abi 7500 sequence detection system

Qiagen DNeasy Blood and Tissue kit (Qiagen, Germantown, MD) was used to extract genomic DNA from frozen muscle and tissue following the manufacturer’s protocol. 100ng of genomic DNA was assayed by qPCR using primers specific to the AAV vector genome: Forward 5’- CCT CAG TGG ATG TTG CCT TTA -3’, Probe 5’-/56-FAM/AAA GCT GCG/ZEN/GAA TTG TAC CCG C/3IABkFQ/-3’ and Reverse 5’- ATG CCA AGT CCT AAG ACT AAA AC -3’. The pAAV.MHCK7.GALGT2(canine) plasmid was cut with SwaI and purified for use as a linear DNA standard. This standard was measured between 50 and 5 million copies in logarithmic increments to generate a linear calibration, for which the Pearson correlation coefficient always equaled or exceeded 0.98. qPCR measures were performed on a TaqMan ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA). Each sample was run in triplicate and the values were averaged. Some samples were spiked with 100 copies of standard and measured to insure no quenching of signal by genomic DNA or by contaminants from the extraction procedure.
To calculate vg/nucleus, the number of vector genomes per μg of genomic canine DNA was divided by 1.8x10⁵ vg/nucleus, assuming 5.4 pg of genomic DNA per diploid nucleus.

Gastrocnemius muscles were homogenized in TRIzol (Invitrogen) and total RNA was extracted and purified using either RNeasy Mini or Micro kits (Qiagen) according to the manufacturer’s instructions. The quality of RNA was determined by electrophoresis using 6000 Nano LabChip kit on Bioanalyzer 2100 (Agilent). The samples without RNA degradation were used to synthesize cDNA using High Capacity Reverse Transcriptase kit (Applied Biosystems). Taqman ABI 7500 sequence detection system (Applied Biosystems) was used for real-time qPCR measurements on the samples using the delta-delta CT method [64 (link)]. qRT-PCR probes for Galgt1 and 18S RNA have been described in previous publications [32 (link),65 (link)]. Primers for dystrophin (Mm01216926_m1), Pax7 (Mm00834079_m1), Myf5 (Mm00435125_m1), MyoD (Mm00440387_m1), Myogenin (Mm00446194_m1), and Myh3 (Mm01332463_m1) were purchased from Applied Biosystems. 18S was used as an internal control. Measures shown are replicates of three to four muscles per condition.