Gavo-cel detection by qPCR in peripheral blood
BLOOD
PBMCs were isolated from whole blood by density gradient centrifugation and cryopreserved as dry pellets. gDNA was extracted from PBMCs using the Qiagen DNA Blood Mini Kit. A duplex real-time PCR assay was performed using TaqMan probes and primers to amplify the oPRE sequence in the TRuC transgene in tandem with endogenous albumin. oPRE copy number (copies per µg of gDNA) was calculated against standard curves consisting of known concentrations of linear oPRE and albumin double-stranded DNA (dsDNA) fragments. All qPCR reactions were performed using the Applied Biosystems ViiA7 Real Time PCR System (Life Technologies). dsDNA used for standard and control material was synthesized using GeneArt Gene Synthesis (Thermo Fisher Scientific), and the sequences are shown below. The sequence used for albumin was derived from within exon 7 of human albumin, National Center for Biotechnology Information reference sequence AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCTGTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCAlbumin dsDNA: 350-bp linear fragmentTTATTTCTGTATGTCCATTTTGAATTTTCTTATGAGAAATAGTATTTGCCTAGTGTTTTCATATAAAATATCGCATGATAATACCATTTTGATTGGCGATTTTCTTTTTAGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGTAAAGAGTCGTCGATATGCTTTTTGGTAGCTTGCATGCTCAAGTTGGTAGAATGGATGCGTTTGGTATCATTGGTGATAGCTGACAGTGGGTTGAGATTGTCTTCTGT