SUMMARY: DNA samples were extracted from ethanol and formalin-fixed decapod crustacean tissue using a new method based on Tetramethylsilane (TMS)-Chelex. It is shown that neither an indigestible matrix of cross-linked protein nor soluble PCR inhibitors impede PCR success when dealing with formalin-fixed material. Instead, amplification success from formalin-fixed tissue appears to depend on the presence of unmodified DNA in the extracted sample. A staining method that facilitates the targeting of samples with a high content of unmodified DNA is provided.Keywords: tetramethylsilane, ethidium bromide, formalin, Carcinus, Lithodidae. RESUMEN: Extracción de ADN a partir de tejido fijado en formol: nueva luz desde el mar abisal. -Muestras de ADN de distintos crustáceos decápodos fueron obtenidas independientemente a partir de tejidos fijados en etanol y tejidos fijados en formol mediante un nuevo protocolo basado en el Tetrametilsilano (TMS)-Chelex. Los resultados obtenidos muestran que el ADN no se encuentra atrapado de forma irreversible en una matriz proteica y que el éxito de amplificación no depende de la extracción de inhibidores de PCR solubles. Sin embargo, nuestros resultados indican que el éxito de amplificación depende de la presencia de ADN no modificado en la muestra. Se incluye un sencillo método de tinción que facilita la identificación de muestras con un alto contenido en ADN no modificado.Palabras clave: tetrametilsilano, bromuro de etidio, formol, Carcinus, Lithodidae.
Introduction: Clinical, biodiversity, and environmental biobanks share many data standards, but there is a lack of harmonization on how data are defined and used among biobank fields. This article reports the outcome of an interactive, multidisciplinary session at a meeting of the European, Middle Eastern, and African Society for Biopreservation and Biobanking (ESBB) designed to encourage a ‘learning-from-each-other’ approach to achieve consensus on data needs and data management across biobank communities.Materials, Methods, and Results: The Enviro-Bio and ESBBperanto Working Groups of the ESBB co-organized an interactive session at the 2013 conference (Verona, Italy), presenting data associated with biobanking processes, using examples from across different fields. One-hundred-sixty (160) diverse biobank participants were provided electronic voting devices with real-time screen display of responses to questions posed during the session. The importance of data standards and robust data management was recognized across the conference cohort, along with the need to raise awareness about these issues within and across different biobank sectors.Discussion and Conclusion: While interactive sessions require a commitment of time and resources, and must be carefully coordinated for consistency and continuity, they stimulate the audience to be pro-active and direct the course of the session. This effective method was used to gauge opinions about significant topics across different biobanking communities. The votes revealed the need to: (a) educate biobanks in the use of data management tools and standards, and (b) encourage a more cohesive approach for how data and samples are tracked, exchanged, and standardized across biobanking communities. Recommendations for future interactive sessions are presented based on lessons learned.
Cyclotrichiids are of ecological and evolutionary interest by virtue of their importance in red tide formation, their highly divergent small subunit (SSU) ribosomal RNA (rRNA) genes, kleptoplastidy, and utility as indicators of eutrophication. However, only seven strains have had their SSU rRNA genes sequenced and their environmental diversity and distribution are largely unknown. We probed 67 globally dispersed freshwater column/sediment and soil DNA samples (eDNAs) and constructed 24 environmental gene libraries using polymerase chain reaction primers specific to an uncharacterised cyclotrichiid subgroup. We reveal a novel, globally ubiquitous freshwater clade comprising 25 genetically distinct SSU ribosomal DNA (rDNA) sequences (SSU-types). Some identical SSU-types were detected at globally widely distributed sites. The SSU-types form four distinct phylogenetic clusters according to marine or non-marine provenance, suggesting at least one major marine-freshwater evolutionary transition within the cyclotrichiids. We used the same primers to sample intensively 18 sampling points in 13 closely situated lakes, each characterised by 14 environmental variables, and showed that molecular detection or non-detection of cyclotrichiids was most significantly influenced by levels of total phosphorus, dissolved organic carbon, and chlorophyll a. Within the subset of lakes in which cyclotrichiids were detected, closely related SSU-types differed in their ecological preferences to pH, total phosphorus, and sample depth.
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