title: Quantification of DNA in urinary porcine bladder matrix using the ACTB gene.
abstract: Extracellular matrix (ECM) is a rich network of proteins and proteoglycans that has proved to be very useful in tissue regeneration. Porcine ECM has been proposed as a biological scaffold, and urinary bladder matrix (UBM) has demonstrated superior biological properties; however, its use in human treatment requires ensuring that it is DNA free. Several protocols have been used for decellularization and to demonstrate the absence of DNA, but until now, a porcine housekeeping gene for quantifying DNA by real-time quantitative PCR (qPCR) has been limiting. The aim of this study was to propose a protocol to quantify the DNA content of decellularized UBM by qPCR for the beta-actin gene (ACTB). A total of 20 porcine bladders were used, and each bladder was divided into three pieces: one as a control and the others decellularized with either SDS or Triton X-100 detergent. The presence of DNA was assessed by histology, spectrophotometry, conventional PCR, and qPCR for the ACTB. Histological analysis demonstrated the absence of nuclei using both protocols. Spectrophotometrical evaluation resulted in DNA concentrations of 1561.4 ± 357.1 and 1211.9 ± 635.2 ng of DNA/mg dry weight after the SDS and Triton X-100 protocols, respectively. DNA was not detected in any protocol by conventional PCR. In contrast, using qPCR, we found 3.9 ± 2.8 ng of DNA/mg dry weight in the Triton X-100 protocol. Therefore, the use of qPCR is a reliable method to quantify residual DNA content after decellularization procedures.
authors.affiliation: Institutional Doctorate in Materials Science and Engineering, Autonomous University of San Luis Potosi, San Luis Potosi, S.L.P., México.
authors.forename: Erika
authors.initials: E
authors.lastname: Silva-Benítez
authors.affiliation: Institutional Doctorate in Materials Science and Engineering, Autonomous University of San Luis Potosi, San Luis Potosi, S.L.P., México.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Soto-Sáinz
authors.affiliation: Basic Science Laboratory, Faculty of Dentistry, Autonomous University of San Luis Potosi, San Luis Potosi, S.L.P., México.
authors.forename: Amaury
authors.initials: A
authors.lastname: Pozos-Guillen
authors.affiliation: Faculty of Biological and Chemical Sciences, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.|Faculty of Dentistry, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.
authors.forename: José Geovanni
authors.initials: JG
authors.lastname: Romero-Quintana
authors.affiliation: Faculty of Biological and Chemical Sciences, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.|Faculty of Dentistry, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.
authors.forename: Maribel
authors.initials: M
authors.lastname: Aguilar-Medina
authors.affiliation: Faculty of Dentistry, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ayala-Ham
authors.affiliation: Pediatric Hospital of Sinaloa, Culiacan, Sinaloa, México.
authors.forename: Eri
authors.initials: E
authors.lastname: Peña-Martínez
authors.affiliation: Faculty of Biological and Chemical Sciences, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.|Faculty of Dentistry, Autonomous University of Sinaloa, Culiacan, Sinaloa, México.
authors.forename: Rosalío
authors.initials: R
authors.lastname: Ramos-Payán
authors.affiliation: Basic Science Laboratory, Faculty of Dentistry, Autonomous University of San Luis Potosi, San Luis Potosi, S.L.P., México. heflores@uaslp.mx.
authors.forename: Héctor
authors.initials: H
authors.lastname: Flores
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D004247:DNA / Q000032:analysis*; D003902:Detergents / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology*; D006801:Humans; D017830:Octoxynol / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013053:Spectrophotometry; D013552:Swine; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds; D001743:Urinary Bladder / Q000166:cytology*
chemical_list: D000199:Actins; D003902:Detergents; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol; D004247:DNA
keywords: Beta-actin gene; Decellularization; Extracellular matrix; Real-time qPCR