Chromosome Microdissection on Semi-Archived Material

Autor(es)

Hamid Al-Rikabi, Ahmed B. ^[1] ; Cioffi, Marcelo de Bello ^{[2, 1]} ; Liehr, Thomas ^[1]

Afiliação do(s) autor(es)

^[1] Friedrich Schiller Univ, Inst Human Genet, Jena Univ Hosp, Jena - Germany

^[2] Univ Fed Sao Carlos, Dept Genet & Evolucao, Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Cytometry Part A; SEP 2019.
• Citações Web of Science: 0

Resumo

Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a similar to 2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20 degrees C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20 degrees C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. (c) 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. (AU)

• Processo FAPESP: 18/22033-1 - Uma abordagem intercontinental para a investigação da evolução cromossômica, diversidade genética e biogeografia na ordem Osteoglossiformes (Teleostei: Osteoglossomorpha). Parte III.
• Beneficiário: Marcelo de Bello Cioffi
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/10240-0 - Diversidade genética, biogeografia e evolução de peixes da ordem Osteoglossiformes (Teleostei, Osteoglossomorpha)
• Beneficiário: Manolo Fernandez Perez
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado