Chlorophyll fluorescence--a practical guide

Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. This review aims to provide an introduction for the novice into the methodology and applications of chlorophyll fluorescence. After a brief introduction into the theoretical background of the technique, the methodology and some of the technical pitfalls that can be encountered are explained. A selection of examples is then used to illustrate the types of information that fluorescence can provide.     

Mammalian RNAi: a practical guide

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Caenorhabditis elegans and Drosophila melanogaster genomes. Recent advances in the design and delivery of targeting molecules now permit efficient and highly specific gene silencing in mammalian systems as well. RNAi offers a simple, fast, and cost-effective alternative to existing gene targeting technologies both in cell-based and in vivo settings. Synthetic small interfering RNA (siRNA) and retroviral short hairpin RNA (shRNA) libraries targeting thousands of human and mouse genes are publicly available for high-throughput genetic screens, and knockdown animals can be rapidly generated by lentivirus-mediated transgenesis. RNAi also holds great promise as a novel therapeutic approach. This review provides insight into the current gene silencing techniques in mammalian systems.     

A practical guide to molecular dating

Molecular dating has now become a common tool for many biologists and considerable methodological improvements have been made over the last few years. However, the practice of estimating divergence times using molecular data is highly variable among researchers and it is not straightforward for a newcomer to the field to know how to start. Here I provide a brief overview of the current state-of-the-art of molecular dating practice. I review some of the important choices that must be made when conducting a divergence time analysis, including how to select and use calibrations and which relaxed clock model and program to use, with a focus on some practical aspects. I then provide some guidelines for the interpretation of results and briefly review some alternatives to molecular dating for obtaining divergence times. Last, I present some promising developments for the future of the field, related to the improvement of the calibration process.     

A practical guide to single-molecule FRET

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.     

A practical guide to measuring local adaptation

Patterns of local adaptation are expected to emerge when selection is spatially heterogeneous and sufficiently strong relative to the action of other evolutionary forces. The observation of local adaptation thus provides important insight into evolutionary processes and the adaptive divergence of populations. The detection of local adaptation, however, suffers from several conceptual, statistical and methodological issues. Here, we provide practical recommendations regarding (1) the definition of local adaptation, (2) the analysis of transplant experiments and (3) the optimisation of the experimental design of local adaptation studies. Together, these recommendations provide a unified approach for measuring local adaptation and understanding the adaptive divergence of populations in a wide range of biological systems.     

Multilocus enzyme electrophoresis: a practical guide

Multilocus enzyme electrophoresis (MEE) uses the relative electrophoretic mobilities of intracellular enzymes to characterize and differentiate organisms by generating an electromorph type (ET). This article presents the chemical conditions that may be useful, a guide to the successful practice of the electrophoretic technique, and analysis of the results.     

A practical guide to methods of parentage analysis

The use of molecular techniques for parentage analysis has been a booming science for over a decade. The most important technological breakthrough was the introduction of microsatellite markers to molecular ecology, an advance that was accompanied by a proliferation and refinement of statistical techniques for the analysis of parentage data. Over the last several years, we have seen steady progress in a number of areas related to parentage analysis, and the prospects for successful studies continue to improve. Here, we provide an updated guide for scientists interested in embarking on parentage analysis in natural or artificial populations of organisms, with a particular focus on computer software packages that implement various methods of analysis. Our survey of the literature shows that there are a few established methods that perform extremely well in the analysis of most types of parentage studies. However, particular experimental designs or study systems can benefit from some of the less well-known computer packages available. Overall, we find that parentage analysis is feasible and satisfying in most systems, and we try to provide a simple roadmap to help other scientists navigate the confusing topography of statistical techniques.     

A practical guide to DNA metabarcoding for entomological ecologists

1. DNA metabarcoding is a cost-effective species identification approach with great potential to assist entomological ecologists. This review presents a practical guide to help entomological ecologists design their own DNA metabarcoding studies and ensure that sound ecological conclusions can be obtained. 2. The review considers approaches to field sampling, laboratory work, and bioinformatic analyses, with the aim of providing the background knowledge needed to make decisions at each step of a DNA metabarcoding workflow. 3. Although most conventional sampling methods can be adapted to DNA metabarcoding, this review highlights techniques that will ensure suitable DNA preservation during field sampling and laboratory storage. The review also calls for a greater understanding of the occurrence, transportation, and deposition of environmental DNA when applying DNA metabarcoding approaches for different ecosystems. 4. Accurate species detection with DNA metabarcoding needs to consider biases introduced during DNA extraction and PCR amplification, cross-contamination resulting from inappropriate amplicon library preparation, and downstream bioinformatic analyses. Quantifying species abundance with DNA metabarcoding is in its infancy, yet recent studies demonstrate promise for estimating relative species abundance from DNA sequencing reads. 5. Given that bioinformatics is one of the biggest hurdles for researchers new to DNA metabarcoding, several useful graphical user interface programs are recommended for sequence data processing, and the application of emerging sequencing technologies is discussed.     

A practical guide to evaluating colocalization in biological microscopy

Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.     

A practical guide to the synthesis of dinitroindolinyl-caged neurotransmitters

This protocol describes a method for efficient chemical synthesis of dinitroindolinyl derivatives of glutamate and γ-aminobutyric acid. These caged neurotransmitters are currently the most chemically and photochemically efficient probes for two-photon photolysis in living brain slices. The protocol only requires basic organic synthesis equipment, and no silica gel column chromatography or NMR spectroscopy is needed at any stage. HPLC is used to purify the caged transmitters at the end of the syntheses. Thus, the synthesis of dinitroindolinyl-caged neurotransmitters is within the scope of a modestly equipped chemistry laboratory.     

A practical guide to the study of social relationships

Behavioral ecologists have devoted considerable effort to identifying the sources of variation in individual reproductive success. 1 - 5 Much of this work has focused on the characteristics of individuals, such as their sex and rank. However, many animals live in stable social groups and the fitness of individuals depends at least in part on the outcome of their interactions with other group members. For example, in many primate species, high dominance rank enhances access to resources and reproductive success. 4 , 5 The ability to acquire and maintain high rank often depends on the availability and effectiveness of coalitionary support. 6 Allies may be cultivated and coalitions may be reinforced by affiliative interactions such as grooming, food sharing, and tolerance. 7 , 8 These findings suggest that if we want to understand the selective pressures that shape the social behavior of primates, it will be profitable to broaden our focus from the characteristics of individuals to the properties of the relationships that they form with others. The goal of this paper is to discuss a set of methods that can be used to quantify the properties of social relationships.