Springer Science and Business Media LLC

Abstract Background As real-time quantitative PCR (RT-QPCR) is increasingly being relied upon for the enforcement of legislation and regulations dependent upon the trace detection of DNA, focus has increased on the quality issues related to the technique. Recent work has focused on the identification of factors that contribute towards significant measurement uncertainty in the real-time quantitative PCR technique, through investigation of the experimental design and operating procedure. However, measurement uncertainty contributions made during the data analysis procedure have not been studied in detail. This paper presents two additional approaches for standardising data analysis through the novel application of statistical methods to RT-QPCR, in order to minimise potential uncertainty in results. Results Experimental data was generated in order to develop the two aspects of data handling and analysis that can contribute towards measurement uncertainty in results. This paper describes preliminary aspects in standardising data through the application of statistical techniques to the area of RT-QPCR. The first aspect concerns the statistical identification and subsequent handling of outlying values arising from RT-QPCR, and discusses the implementation of ISO guidelines in relation to acceptance or rejection of outlying values. The second aspect relates to the development of an objective statistical test for the comparison of calibration curves. Conclusion The preliminary statistical tests for outlying values and comparisons between calibration curves can be applied using basic functions found in standard spreadsheet software. These two aspects emphasise that the comparability of results arising from RT-QPCR needs further refinement and development at the data-handling phase. The implementation of standardised approaches to data analysis should further help minimise variation due to subjective judgements. The aspects described in this paper will help contribute towards the development of a set of best practice guidelines regarding standardising handling and interpretation of data arising from RT-QPCR experiments.

Abstract Background Grasshopper serves as important model system in neuroscience, development and evolution. Representatives of this primitive insect group are also highly relevant targets of pest control efforts. Unfortunately, the lack of genetics or gene specific molecular manipulation imposes major limitations to the study of grasshopper biology. Results We investigated whether juvenile instars of the grasshopper species Schistocerca americana are conducive to gene silencing via the systemic RNAi pathway. Injection of dsRNA corresponding to the eye colour gene vermilion into first instar nymphs triggered suppression of ommochrome formation in the eye lasting through two instars equivalent to 10–14 days in absolute time. QRT-PCR analysis revealed a two fold decrease of target transcript levels in affected animals. Control injections of EGFP dsRNA did not result in detectable phenotypic changes. RT-PCR and in situ hybridization detected ubiquitous expression of the grasshopper homolog of the dsRNA channel protein gene sid-1 in embryos, nymphs and adults. Conclusion Our results demonstrate that systemic dsRNA application elicits specific and long-term gene silencing in juvenile grasshopper instars. The conservation of systemic RNAi in the grasshopper suggests that this pathway can be exploited for gene specific manipulation of juvenile and adult instars in a wide range of primitive insects.

Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries. Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM). The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification. The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.

Abstract Background The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. Results Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. Conclusion Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course. Incubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold. Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.

Abstract Background Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). Results Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. Conclusion We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.