Avian DNA extraction: An economical and efficient alternative for Farmer-fixed samples
DOI:
https://doi.org/10.36253/caryologia-3420Keywords:
DNA extraction, cell preservation, cytogenomics, sustainable methods, 3Rs principlesAbstract
Cytogenetics laboratories often accumulate vast collections of cells fixed in Farmer’s solution (3 parts methanol to 1-part glacial acetic acid), stored long-term in freezers. While many of these samples are unsuitable for conventional cytogenetic analyses, they hold potential for molecular applications, especially as ethical restrictions around the collection of biological material through invasive procedures (e.g., biopsies, tissue excision, bone marrow aspiration) become increasingly stringent. However, extracting DNA from these cells presents significant challenges, such as structural fragility induced by the fixative and potential genetic material degradation, which can compromise subsequent analyses, including PCR. This study developed and standardized a protocol for extracting DNA from Farmer-fixed avian cells using accessible and low-cost reagents. The method proved economical and efficient, even for decades-old samples, recovering DNA suitable for cytogenomic and molecular studies. This approach significantly advances sustainable practices in science by utilizing long-stored samples that might otherwise be discarded, this approach provides a cost-effective strategy that reduces the need for new collections and aligns with current ethical guidelines in molecular genetics research. Compared to commercial kits, the protocol demonstrated economic viability while expanding the use of biological collections in genetic research and evolutionary studies.
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References
1. Tan SC, Yiap BC (2009). DNA, RNA, and protein extraction: The past and the present. J Biomed Biotechnol 2009:1–10.
2. Lott JA, Coleman WB, Tsongalis GJ, eds. (1997). Clinical Pathology of Pancreatic Disease and Molecular Diagnostics: For the Clinical Laboratorian. 1st ed. [s.l.]: [s.n.].
3. Amorim MR, Vargas FR, Llerena Junior JC, Pombo-de-Oliveira MS (2007). DNA extraction from fixed cytogenetics cell suspensions. Genet Mol Res 6(3):500–503.
4. Schrader C, Schielke A, Ellerbroek L, Johne R (2012). PCR inhibitors – occurrence, properties and removal. J Appl Microbiol 113(5):1014–1026.
5. Floridia M, Pafundi S, Cecchi C, et al. (2023). Comparative effects of fixatives on RNA and DNA integrity: Implications for molecular diagnostics. Diagn Mol Pathol.
6. Pereira MA, Dias AR, Faraj SF, Cirqueira CS, Tomitao MT, Nahas SC, Ribeiro U Jr, de Mello ES (2015). Histopathology 66:388–397. https://doi.org/10.1111/his.12532.
7. Díaz C, Carmona E, Nuñez R, et al. (2020). Ethics in biological sample preservation: Applications and challenges in wildlife research. Biol Conserv 250:108–120.
8. Díaz L, Zambrano E, Flores ME, et al. (2020). Ethical considerations in animal research: The principle of 3R’s. Rev Invest Clin 73(4):199–209. https://doi.org/10.24875/RIC.20000380.
9. Hubrecht R, Carter E (2019). The 3Rs and the ethics of animal research. Anim Res Ethics 12(3):45–58.
10. Kulkarni MD, Shringarpure R, Bhatt P, Prakash N, Prakash V (2015). A simple and inexpensive protocol for DNA isolation from avian blood. J Bombay Nat Hist Soc 112:3–7. https://doi.org/10.17087/jbnhs/2015/v112i1/92186.
11. Srinivasan M, Sedmak D, Jewell S (2002). Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161(6):1961–1971. https://doi.org/10.1016/S0002-9440(10)64472-0.
12. Miyaki CY (2001). Genética e evolução aplicada à conservação. In: Ornitologia e Conservação. Da Ciência às Estratégias. Editora Unisul, pp. 239–246.
13. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613–616. https://doi.org/10.1016/0014-4827(60)90138-5.
14. Garnero AV, Gunski RJ (2000). Comparative analysis of the karyotype of Nothura maculosa and Rynchotus rufescens (Aves: Tinamidae): A case of chromosomal polymorphism. Nucleus 43:64–70.
15. Nogueira DM, Freitas AAR (2013). Fixed cytogenetic cells suspension: An alternative for obtaining DNA of birds. Rev Bras Ornitol 21:120–123.
16. Grimm H, Biller-Andorno N, Buch T, et al. (2023). Advancing the 3Rs: innovation, implementation, ethics and society. Front Vet Sci 10:1185706. https://doi.org/10.3389/fvets.2023.1185706.
17. Kalkan K, Kekecoglu M (2024). Ethical principles and rules in experimental animal studies: A comprehensive review. Düzce Tıp Fakültesi Dergisi 26. https://doi.org/10.18678/dtfd.1497730.
18. Beránek M, Hegerová J, Michajlíková M, Dulíček P, Palicka V (2022). No inhibitory effect of heparinized blood on real-time PCR analysis of thrombophilic mutations. Folia Biol (Praha) 68:201–205. https://doi.org/10.14712/fb2022068050201.
19. Floridia V, Sfulcini M, D’Alessandro E, et al. (2023). Effect of different anticoagulant agents on immune-related genes in leukocytes isolated from the whole-blood of Holstein cows. Genes (Basel) 14(2):406. https://doi.org/10.3390/genes14020406.
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