Publicação
Optimization of DNA and RNA extraction protocols for Phaeodactylum tricornutum towards allowing downstream untargeted epigenomic and transcriptomic analysis
| Resumo: | Epigenetics studies the variations in phenotype that are not caused by changes in the DNA sequence. Understanding how contaminants can influence the epigenome, and consequently the transcriptome and the phenotype of model organisms such as Phaeodactylum tricornutum is the aim of the research where this work is included. In the present work, the focus was to optimize the extraction of DNA and RNA from P. tricornutum, enabling the planned epigenome and transcriptome analysis with this organism following exposure to model contaminants. P. tricornutum, a unicellular marine diatom, is a widely studied model organism in the field of algal biology due to its unique characteristics. In this way, the culturing of the diatom was learnt, and cultures were grown to generate biomass for several extraction trials that relied on different techniques and/or kits. Success of the trials was examined through DNA/RNA yield and quality control techniques, namely generalist spectrophotometric quantification, specific fluorimetry-based quantification and agarose-gel electrophoresis. The results showed that the isopropanol precipitation procedure was the most efficient for DNA extraction. Regarding RNA extraction, a Qiagen kit had the best outcome. Still, further work is required to fine-tune the extraction protocols towards reaching the demanding yields and quality standards required for whole genome and whole transcriptome sequencing techniques. |
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| Autores principais: | Fonseca, Larissa |
| Assunto: | Phaeodactylum tricornutum DNA extraction RNA extraction |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | relatório |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | Epigenetics studies the variations in phenotype that are not caused by changes in the DNA sequence. Understanding how contaminants can influence the epigenome, and consequently the transcriptome and the phenotype of model organisms such as Phaeodactylum tricornutum is the aim of the research where this work is included. In the present work, the focus was to optimize the extraction of DNA and RNA from P. tricornutum, enabling the planned epigenome and transcriptome analysis with this organism following exposure to model contaminants. P. tricornutum, a unicellular marine diatom, is a widely studied model organism in the field of algal biology due to its unique characteristics. In this way, the culturing of the diatom was learnt, and cultures were grown to generate biomass for several extraction trials that relied on different techniques and/or kits. Success of the trials was examined through DNA/RNA yield and quality control techniques, namely generalist spectrophotometric quantification, specific fluorimetry-based quantification and agarose-gel electrophoresis. The results showed that the isopropanol precipitation procedure was the most efficient for DNA extraction. Regarding RNA extraction, a Qiagen kit had the best outcome. Still, further work is required to fine-tune the extraction protocols towards reaching the demanding yields and quality standards required for whole genome and whole transcriptome sequencing techniques. |
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