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Extraction of Nucleic Acids from Ancient Formalin- and Ethanol-Preserved Specimens of the Tapeworm Bertiella studeri : Which Method Works Best?

Extraction of Nucleic Acids from Ancient Formalin- and Ethanol-Preserved Specimens of the... Abstract: In this study we show that intact DNA can be recovered from both alcohol- and formalin-preserved specimens of the parasitic tapeworm Bertiella studeri for >1 yr after the original fixation and can be successfully amplified and quantified using real-time polymerase chain reaction (RT-PCR). Three different DNA extraction techniques on formalin- and alcohol-fixed material were evaluated to determine which is best suited for obtaining DNA of sufficient yield and purity to be used in downstream applications such as RT-PCR. These techniques included a first organic extraction procedure with an extensive washing step based on a glycine-containing buffer, a second organic extraction procedure that omits the glycine-containing buffer and halves the number of washes in organic solvents, and a third procedure that involves the use of a silica-based DNA binding column from Qiagen © . The quality of extracted DNA was first examined by agarose gel electrophoresis and ethidium bromide staining and the concentrations were evaluated by OD 260 . We then used an RT-PCR Applied Biosystems TaqMan® Fluorogenic 5′ Nuclease Gene Expression Assay based on the detection of a universal eukaryotic 18S rRNA gene sequence to evaluate the efficiency of the 3 methods. Here we report that, first, the use of a silica column-based DNA extraction technique results in the maximum yield of DNA, on average 55% higher than for the organic extraction methods; second, the use of glycine as a formaldehyde-binding agent in the washing buffer does not necessarily result in a better DNA yield and; third, specimens preserved in ethanol result in significantly higher yields of amplifiable DNA than do specimens preserved in formalin. We, therefore, strongly recommend the use of ethanol as the appropriate fixative if specimens are to be used for molecular work. This is the first report of DNA extraction from preserved specimens of Bertiella studeri . http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Parasitology Allen Press

Extraction of Nucleic Acids from Ancient Formalin- and Ethanol-Preserved Specimens of the Tapeworm Bertiella studeri : Which Method Works Best?

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Publisher
Allen Press
Copyright
Copyright © American Society of Parasitologists
Subject
ECOLOGY-EPIDEMIOLOGY-BEHAVIOR
ISSN
0022-3395
eISSN
1937-2345
DOI
10.1645/GE-3197.1
pmid
23234306
Publisher site
See Article on Publisher Site

Abstract

Abstract: In this study we show that intact DNA can be recovered from both alcohol- and formalin-preserved specimens of the parasitic tapeworm Bertiella studeri for >1 yr after the original fixation and can be successfully amplified and quantified using real-time polymerase chain reaction (RT-PCR). Three different DNA extraction techniques on formalin- and alcohol-fixed material were evaluated to determine which is best suited for obtaining DNA of sufficient yield and purity to be used in downstream applications such as RT-PCR. These techniques included a first organic extraction procedure with an extensive washing step based on a glycine-containing buffer, a second organic extraction procedure that omits the glycine-containing buffer and halves the number of washes in organic solvents, and a third procedure that involves the use of a silica-based DNA binding column from Qiagen © . The quality of extracted DNA was first examined by agarose gel electrophoresis and ethidium bromide staining and the concentrations were evaluated by OD 260 . We then used an RT-PCR Applied Biosystems TaqMan® Fluorogenic 5′ Nuclease Gene Expression Assay based on the detection of a universal eukaryotic 18S rRNA gene sequence to evaluate the efficiency of the 3 methods. Here we report that, first, the use of a silica column-based DNA extraction technique results in the maximum yield of DNA, on average 55% higher than for the organic extraction methods; second, the use of glycine as a formaldehyde-binding agent in the washing buffer does not necessarily result in a better DNA yield and; third, specimens preserved in ethanol result in significantly higher yields of amplifiable DNA than do specimens preserved in formalin. We, therefore, strongly recommend the use of ethanol as the appropriate fixative if specimens are to be used for molecular work. This is the first report of DNA extraction from preserved specimens of Bertiella studeri .

Journal

The Journal of Parasitology – Allen Press

Published: Jun 1, 2013

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