Evaluation of three DNA extraction protocols for forensic STR typing after laser capture microdissection.
ABSTRACT In forensic sciences, short tandem repeat (STR) analysis is a valuable tool in identifying the donor(s) of biological stains. Laser capture microdissection (LCM) can be used as a cell separating technique to isolate specific cell types in mixed samples. An important challenge lies in the development of a DNA isolation method appropriate for laser microdissected cells, as these samples usually contain minute amounts of cells. In this study three different DNA isolation methods for LCM collected cells were compared. The PicoPure DNA extraction method outperformed both other methods (IQ™ system and short alkaline method). Consequently, the minimal number of LCM collected cells necessary for STR typing was determined. Using the PicoPure DNA extraction method, full DNA profiles could be obtained from as little as 10 cells. Nevertheless, despite the occurrence of allelic drop out in some of the samples, lower amounts of cells gave rise to useful DNA profiles.
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ABSTRACT: TO THE EDITOR: Although 2 major diseases of amphibians, chytridiomycosis and ranavirosis, have been relatively well studied, enigmatic amphibian disease and death not attributable to any of the known amphibian diseases frequently occur (1). We describe an apparently new disease in salamanders that is associated with a novel genus within the family Chlamydiaceae.Emerging Infectious Diseases 06/2012; 18(6):1020-2. · 6.79 Impact Factor
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ABSTRACT: Knowledge concerning microbial infectious diseases in the current amphibian crisis is rudimentary and largely limited to ranavirosis and chytridiomycosis. The family Chlamydiaceae is gaining attention as a common cause of disease in amphibians and may harbour new and emerging amphibian pathogens. We identified a novel species of Chlamydiales (Candidatus Amphibiichlamydia ranarum) with a prevalence of 71% in exotic invasive bullfrog tadpoles (Lithobates catesbeianus) from an introduced population in the Netherlands. The sequence of a 1474 bp 16S rRNA gene fragment showed that the novel taxon forms a well-defined clade with 'Candidatus Amphibiichlamydia salamandrae' within the Chlamydiaceae family. Although none of the tadpoles examined showed signs of clinical disease, urgent evaluation of its pathogenic potential for native amphibian species is required.Environmental Microbiology Reports 02/2013; 5(1):105-8. · 2.71 Impact Factor
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ABSTRACT: Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation are however crucial for the successfulness of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one system based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user friendliness to sample quality. The comparison was performed on two types of samples, i.e. peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. The UV system not only allows faster and more precise sample collection, the obtained samples, even single cell samples, can also be used for DNA extraction and downstream PCR applications. RNA based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for RT-qPCR analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays.Analytical Biochemistry 04/2013; · 2.58 Impact Factor