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High-resolution insight into parasitic infections and diet of past populations in Northern Europe and the Middle East (500 BC- 1700 AD) was obtained by pre-concentration of parasite eggs from ancient latrines and deposits followed by shotgun sequencing of DNA. Complementary profiling of parasite, vertebrate and plant DNA proved highly informative in the study of ancient health, human-animal interactions as well as animal and plant dietary components. Most prominent were finding of soil-borne parasites transmitted directly between humans, but also meat-borne parasites that require consumption of raw or undercooked fish and pork. The detection of parasites for which sheep, horse, dog, pig, and rodents serves as definitive hosts are clear markers of domestic and synanthropic animals living in closer proximity of the respective sites. Finally, the reconstruction of full mitochondrial parasite genomes from whipworm (Ascaris lumbricoides) and roundworm species (Trichuris trichiura and Trichuris muris) and estimates of haplotype frequencies elucidates the genetic diversity and provides insights into epidemiology and parasite biology. [ABSTRACT FROM AUTHOR]

The article presents studies which examined highly variable nuclear markers in worm isolates of human and pig origin from many different geographic locations. The studies compared Ascaris isolates from pigs and humans and have identified close similarities in mitochondrial and single nuclear markers and 18S rRNA. The capability of next-generation sequencing to offer whole genome sequences of multiple individuals as exemplified for Trichinella and Schistosoma worms is tackled.

RT-isoPCR provides multiplex amplification of mRNA targets using a first-stage multiplex RT-PCR reaction with subsequent isothermal amplification for individual target loci detection. We demonstrate detection of 24 mRNA targets with high specificity and sensitivity without compromising sample variation or introducing biases between targets. [ABSTRACT FROM AUTHOR]

Background: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. Methods/Findings: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. Conclusions: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios. [ABSTRACT FROM AUTHOR]

In situ hybridization is a powerful method to provide information about contextual distribution and cellular origin of nucleic acids, e.g., in formalin-fixed paraffin-embedded (FFPE) samples of tissue. Particularly the recently discovered classes of noncoding RNA (ncRNA) including endo-siRNAs and microRNAs require such a technique to enable their study and visualization in natural contexts, and in the last decade, many advances have been made, increasing our ability to specifically detect small ncRNAs. One of the key developments has been the demonstration of the superiority of using locked nucleic acid (LNA)-modified DNA probes for the detection of ncRNA in tissue. Here, we describe an alternative in situ hybridization protocol employing oligonucleotide probes consisting of combinations of LNA and 2´-O-methyl RNAs that under optimized hybridization buffer conditions can provide a highly sensitive assay performance with only 1 h hybridization time.

A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e.g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in combination or sequentially through the ability of the HistoFlex to monitor assays without disassembly.