Optimization of DNA Extraction from Moth Tissues Across Different Life Stages
Karvonen, Aino (2025)
2025
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Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:amk-2025052616220
https://urn.fi/URN:NBN:fi:amk-2025052616220
Tiivistelmä
Insects are the largest taxonomic group of animals offering a rich foundation for fascinating evolutionary and ecological research. Insect DNA has been studied under many different scopes to understand genetics, ecosystems, populational studies and many other studies. Insect tissue presents unique challenges for DNA extraction mostly due to their small size, chitinous exoskeletons and possible contaminants from digested food or microbes. However, as DNA extraction has become a routine procedure with highly developed techniques and instruments, many of these troubleshooting difficulties can be avoided by choosing the right conditions and techniques.
This thesis work was conducted at the Finnish Museum of Natural History (Luomus), University of Helsinki, in the Luomus DNA laboratory. The purpose was to optimize DNA extraction from ethanol-preserved moth (Lepidoptera) tissue samples using a DNeasy® Blood &Tissue kit for DNA extraction (QIAGEN). The work begun by manually testing out differing extraction methods and tissue types. The tested variables were sample grinding method, lysis incubation time, extraction protocol and final elution volume. After concluding which method was best for the highest quality and quantity, the bulk sample extraction was conducted using an automated instrument QIAcube Connect (QIAGEN). All samples were analyzed with NanoDrop and gel electrophoresis, and some with Qubit, to see and compare results of the extraction.
The results of optimization indicated that percentually the best yield and highest quality was achieved from pupal tissues, with larva following narrowly. Manual grinding with a pestle and a reduced elution volume were the best approaches for extraction. Other conditions were deemed as insignificant in relation to quality and quantity. Purity and concentration were slightly better in the bulk extractions than in the manually processed samples. After this thesis project, the bulk samples will undergo next generation sequencing.
This thesis work was conducted at the Finnish Museum of Natural History (Luomus), University of Helsinki, in the Luomus DNA laboratory. The purpose was to optimize DNA extraction from ethanol-preserved moth (Lepidoptera) tissue samples using a DNeasy® Blood &Tissue kit for DNA extraction (QIAGEN). The work begun by manually testing out differing extraction methods and tissue types. The tested variables were sample grinding method, lysis incubation time, extraction protocol and final elution volume. After concluding which method was best for the highest quality and quantity, the bulk sample extraction was conducted using an automated instrument QIAcube Connect (QIAGEN). All samples were analyzed with NanoDrop and gel electrophoresis, and some with Qubit, to see and compare results of the extraction.
The results of optimization indicated that percentually the best yield and highest quality was achieved from pupal tissues, with larva following narrowly. Manual grinding with a pestle and a reduced elution volume were the best approaches for extraction. Other conditions were deemed as insignificant in relation to quality and quantity. Purity and concentration were slightly better in the bulk extractions than in the manually processed samples. After this thesis project, the bulk samples will undergo next generation sequencing.