Preservation of avian blood and tissue samples for DNA analyses

Abstract

A problem frequently faced by researchers involved in collecting tissues for DNA isolation is the preservation of samples in the field prior to and during their transportation to the laboratory. Prevention of DNA degradation is usually achieved through freezing. As this is not always practical, we have tested the efficiency of chemical solutions containing high concentrations of salts (e.g., NaCl, EDTA, and diaminocyclohexanetetraacetate) and detergent at preserving DNA in bird tissue and blood samples stored at ambient temperature for extended periods of time. For blood samples, we recommend the use of a buffer that lyses the cells and nuclei and contains 0.01 M Tris, 0.01 M NaCl, 0.01 M EDTA, and 1% n-lauroylsarcosine, Ph 7.5. Tissue samples are best preserved as small pieces in a saline solution made of 20% dimethyl sulfoxyde, 0.25 M EDTA, saturated with NaCl, pH 8.0. DNA extracted from samples preserved in these solutions for up to 24 weeks was compared with DNA recovered from tissue samples stored at −70 °C and blood samples stored at −70 and −20 °C. Yields were similar, averaging 300 μg/0.2 g of tissue and 500 μg/50 μL of blood. Quality of DNA in terms of fragment size, ability to be cut by restriction enzymes, and ability to hybridize to radioactive probes was also similar between cryopreserved and chemically preserved samples. Yields of DNA recovered from tissue samples preserved in 70% ethanol for 6 or 11 weeks was very low and significant degradation was observed. We have also examined how DNA contained in crude avian blood samples withstands freeze–thaw cycles. We found normal yields and no significant degradation of DNA in samples that experienced up to six cycles. We encourage field researchers who refrain from preserving tissue samples because of logistical problems, such as transporting liquid nitrogen containers in the field, to consider using these solutions. Both solutions can also facilitate exchanges of samples between laboratories, and they form an alternative to storage of samples at −70 °C for laboratories and museums with limited access to deep-freezing facilities.