title: A reliable and feasible qPCR strategy for titrating AAV vectors.
abstract: BACKGROUND
  Previous studies have revealed that traditional real-time quantitative PCR (qPCR) underestimates adeno-associated virus (AAV) titer. Because the inverted terminal repeat (ITR) exists in all AAV vectors, the only remaining element from the wild genome could form special configurations to interfere with qPCR titration. To solve this problem, a modified and universal qPCR method was tested and established.
  
  
  METHODS
  In this work, there was a great variation in titration of ssAAV2-EGFP (Enhanced Green Fluorescence Protein) and scAAV2-EGFP genome by traditional qPCR. For ssAAV2-EGFP, the highest titer was found by using the targeting EGFP primers and the lowest titer was measured by those targeting bovine growth hormone polyA element (pBGH) primers.
  
  
  RESULTS
  Experimental data were reverse for ssAAV2-EGFP and scAAV2-EGFP. Here we report an improved and universal SmaI qPCR method, based on cleaving all ITRs in AAV2 genome by SmaI with several advantages: (1) impact of all ITRs in ssAAV2 and scAAV2 was dismissed; (2) titers increased remarkably, up to 7-fold, especially for scAAV2; (3) the variation of titers was reduced when different primers were applied. A similar phenomenon was also observed in other ssAAV2 and scAAV2 products when the range of titration was at 3×107 to 7×109 V.G/µl in this study.
  
  
  CONCLUSIONS
  This modified qPCR strategy can increase rAAV' titer and reduce titration variance, possibly become a universal method for titrating AAV vectors.
authors.affiliation: School of Biomedical Science and Institute of Molecular Medicine, Huaqiao University and Engineering Research Center of Molecular Medicine, Ministry of Education, Quanzhou, Fujian, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Xiuling
authors.initials: X
authors.lastname: Cui
authors.forename: Mingxi
authors.initials: M
authors.lastname: Wang
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
authors.forename: Ruian
authors.initials: R
authors.lastname: Xu
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D017931:DNA Primers / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D005240:Feasibility Studies; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D015843:Serpins / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism
chemical_list: D017931:DNA Primers; D015843:Serpins; D053221:TNF-Related Apoptosis-Inducing Ligand; C485184:enhanced green fluorescent protein; C051324:kallistatin; D049452:Green Fluorescent Proteins