Sample collection

Abstract: This laboratory manual consists of 2 sections which describe methods of examination of human semen and semen-cervical mucus interaction in order to standardize procedures and facilitate evaluation and comparison of research reports The section on semen collection and examination discusses and makes recommendations for sample collection and delivery; initial examination; motility assessment estimation of sperm density; examination of particulate debris; agglutination; sperm viability; counting the spermatozoa; and analysis of morphological characteristics of germinal cells including preparation of seminal fluid smears staining method and classification and quantification of germinal cells and leucocytes Photomicrographs are provided to demonstrate morphological characteristics of normal and abnormal mature sperm immature germinal cells and leucocytes and epithelial cells Appendices provide information on frequency of various sperm forms in a normal ejaculate Papanicolaou staining procedure for sperm and the Bryan/Leishman stain for seminal fluid morphology smears A sample record for sperm analysis is also included The section on sperm-cervical mucus interaction describes the composition and characteristics of the mucus the collection procedure storage and preservation and evaluation including pH Methods of evaluating sperm-cervical mucus interaction are then described The timing and techniques of the post-coital test vaginal pool sample exocervical and low cervical samples and endocervical samples and their interpretation are discussed Instructions are provided for in vitro studies including the capillary tube test and the slide technique

Abstract: Summary In conclusion, the eFP Browser is a convenient tool forinterpreting and visualizing gene expression and other data. Notonly is it valuable for its compatibility to existing resources but ithas also been loaded with several useful data sets. The variousmodes and other features allow the user to extract an array ofconclusions and/or generate useful hypotheses. We hope thatmany researchers will be able to use the eFP Browser both tounderstand particular microarray or other experimental results, aswell as to communicate their own findings. MATERIALS AND METHODS The eFP Browser is implemented in Python and makes use of thePython Imaging Library (PIL) Build 1.1.5 (www.python.org),which we modified to provide an optimized flood pixel re-placement function called replaceFill, and other Python modules,as described on the eFP Browser development homepage. Theinputs for the eFP Browser are illustrated in Figure 1. Apictographic representation of the sample collection as a Targa-based image is required, as is an XML control file, shown in detailin Figure 1B. Two other inputs are a database of gene identifiersand their appropriate microarray element lookups and annota-tions, and a database of gene expression values for the givensamples. In the case of the Arabidopsis, Cell and Mouse eFPBrowsers, we have mirrored publicly-available microarray datafrom several sources – described in the Data Sources andsubsequent two sections – in our Bio-Array Resource [10]. Theseinputs are used by the eFP Browser algorithm to generate anoutput image for a user’s gene identifier.The eFP Browser algorithm itself is programmed in an object-oriented manner. The main program, efpWeb.cgi, is responsiblefor the creation of the HTML code for the user interface andpresentation of the output image. It calls on four modules tocomplete the task. These modules are 1) efp.py, which performsmost of the functions for the generation of the output image,including the parsing of the XML control file, average andstandard deviation calculations, fold-change relative to controlvalue calculations, and image map HTML code; 2) efpDb.py,which connects to the gene expression, microarray element andannotation databases, and returns the appropriate values uponbeing called; 3) efpImg.py, which formulates the actual colourreplace calls on the Targa input image; and 4) efpXML.py, whichidentifies the XML control files that are present in the eFPBrowser’s data directory. These are displayed to the user in theData Source drop-down, thus obviating the need to have themhard-coded in the main efpWeb.cgi program.In the case of the Cell eFP Browser, data in the SUBAdatabase indicate the presence of a given protein in a particularsubcellular location, either based on computational methods or asmolecularly documented by mass spectrometric analysis ofsubcellular fractions, GFP fusions etc. [11]. We have used a simpleheuristic to turn these data into a confidence score for a given geneproduct’s presence in a given subcellular compartment:confidence~X

Abstract: The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

Abstract: After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.