title: Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.
abstract: The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.
authors.affiliation: Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Momoko
authors.initials: M
authors.lastname: Nagai
authors.affiliation: Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.|Frontier Biosciences, Osaka University Graduate School, Osaka, Japan.
authors.forename: Katsura
authors.initials: K
authors.lastname: Minegishi
authors.affiliation: Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.|School of dentistry, Aichi Gakuin University, Nagoya, Japan.
authors.forename: Munekazu
authors.initials: M
authors.lastname: Komada
authors.affiliation: Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Tsuchiya
authors.affiliation: Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kameda
authors.affiliation: Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.|Human Health Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Shigehito
authors.identifier: http://orcid.org/0000-0002-8194-6927
authors.initials: S
authors.lastname: Yamada
mesh_terms: D019342:Acetic Acid; D000013:Congenital Abnormalities / Q000175:diagnosis / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D004622:Embryo, Mammalian; D005557:Formaldehyde; D006801:Humans; D010851:Picrates; D016133:Polymerase Chain Reaction; D011309:Preservation, Biological / Q000379:methods; D013997:Time Factors
chemical_list: D010851:Picrates; D005557:Formaldehyde; C026239:Bouin's solution; D004247:DNA; D019342:Acetic Acid
keywords: DNA; extraction; human embryo; long-term fixation