High-throughput, multiparameter analysis of single cells.

Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays.

A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells.

Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties - e.g. low sample dilution, low contamination - that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.

A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray.

A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.

A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.

Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.

Wherefrom and whereabouts of an alien: the American liver fluke Fascioloides magna in Austria: an overview.

The giant liver fluke Fascioloides magna, an invasive species originating from North America, was recorded in Austria in the wild for the first time in 2000. Since then, various data concerning the epidemiology in snail intermediate hosts and cervid final hosts have been reported. Galba truncatula acts as snail intermediate host, and red deer, roe deer and fallow deer act as final hosts. G. truncatula is abundant throughout the region, especially along muddy shores of slow-flowing branches of the river system. Prevalence in deer (20-100 %) is much higher than in snails (0.03-0.2 %). Despite medical treatment of parts of the deer population, the parasite has successfully established itself on both sides of the Danube floodplain environments southeast of Vienna. Genetic analysis revealed that the infection of Austrian deer populations apparently originated from foci in the Czech Republic or from populations of Danube tributaries. Areas adjacent southwards, which will soon be joined through wildlife crossings, have not yet evidenced F. magna. Nonetheless, these environments are inhabited by host snails and deer and therefore constitute suitable habitats for F. magna. Invading alien parasites not only threaten native individual hosts but also influence host populations, thus potentially also modifying parasite communities and interactions. The host range of F. magna includes a variety of potential hosts, notably other Lymnaeidae as potential intermediate hosts and various ungulates, including sheep and cattle, as final hosts. Because eradication after medical treatment was unsuccessful, and due to the risk of further spread of the parasite into unaffected regions, enhanced control strategies need to be developed. We recommend assessment of introduction pathways and dispersal, continuous monitoring of host abundance and distribution and the prevalence of flukes in intermediate and final hosts, as well as coordinated and concerted actions with neighbouring countries. This strategy could help to reduce potential negative impacts of this and other invasive parasites on host populations in Europe.

Recovery of Fascioloides magna (Digenea) population in spite of treatment programme? Screening of Galba truncatula (Gastropoda, Lymnaeidae) from Lower Austria.

During the past decade, Fascioloides magna, the large American liver fluke, has spread within free-living deer in wetlands of the Danube in Lower Austria. The aim of this study was to determine the current infection rates with F. magna and other digenean parasites in the intermediate host snail Galba truncatula from risk areas in Lower Austria. A total of 3444 G. truncatula were collected and examined microscopically for the presence of digenean trematodes. A set of randomly selected snails and isolated trematode stages were also investigated molecular biologically by PCR and sequencing. Digenean parasites were detected with a prevalence of 2.41% (1.83% Paramphistomoidea; 0.46% Echinostomatoidea; 0.09% Strigeida; 0.06% Plagiorchiida). F. magna was found with an overall prevalence of 0.23%, which may indicate a recovery of the parasite population in spite of an ongoing triclabendazole treatment programme. Moreover, high risk areas and a seasonality of infections were observed.