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Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.
Abstract: We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.
Publication Date: 2010-08-17 PubMed ID: 20730710DOI: 10.4238/vol9-3gmr818Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research compared three different methods of extracting DNA from horse peripheral blood mononuclear cells (PBMC) and lung tissues, evaluating aspects like DNA concentration, purity, and cost. DNA extraction results varied based on the protocol and sample type, suggesting that different techniques should be applied accordingly.
Research Methods
- The study included 34 samples taken from PBMC and 33 samples taken from horse lung fragments.
- Three protocols were used on these samples to extract DNA. Protocol A involved phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C applied the DNAzol® reagent kit.
- Criteria used for evaluation included the final DNA concentration, purity of the extracted DNA, percentage of successful PCR amplification using beta-actin, and the cost of each protocol.
Findings
- For DNA extraction from lung fragments, Protocol A emerged as the best method. It produced the highest DNA concentrations and showed 100% sensitivity in PCR amplification. Protocols C and B followed in this order with descending efficiency.
- When it came to PBMC samples, however, Protocol B proved to be the most effective, exhibiting the highest sensitivity in PCR amplification (100%), followed by Protocols C and A.
Conclusions
- The findings demonstrated that the optimal method for DNA extraction depends on the type of sample. Protocol A produced the best results for lung tissue, whereas Protocol B worked best for PBMCs.
- The researchers concluded that, depending on the type of equine sample, different DNA extraction protocols should be applied. For PCR diagnostic purposes, Protocol A is recommended for lung fragment samples and Protocol B for PBMC samples.
Cite This Article
APA
Santos EM, Paula JF, Motta PM, Heinemann MB, Leite RC, Haddad JP, Del Puerto HL, Reis JK.
(2010).
Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.
Genet Mol Res, 9(3), 1591-1598.
https://doi.org/10.4238/vol9-3gmr818 Publication
Researcher Affiliations
- Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil. elmaira27@yahoo.com.br
MeSH Terms
- Animals
- DNA / isolation & purification
- Horses
- Leukocytes, Mononuclear / metabolism
- Lung / metabolism
- Polymerase Chain Reaction
Citations
This article has been cited 12 times.- Mahmmoud EN, Hamad MA, Khudhur ZN. Detection of Mycoplasma gallisepticum in broiler chickens by PCR. Open Vet J 2022 May-Jun;12(3):329-334.
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