Showing papers in "CSH Protocols in 2010"

Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing

Abstract: The large amount of DNA sequence data generated by high-throughput sequencing technologies often allows multiple samples to be sequenced in parallel on a single sequencing run. This is particularly true if subsets of the genome are studied rather than complete genomes. In recent years, target capture from sequencing libraries has largely replaced polymerase chain reaction (PCR) as the preferred method of target enrichment. Parallelizing target capture and sequencing for multiple samples requires the incorporation of sample-specific barcodes into sequencing libraries, which is necessary to trace back the sample source of each sequence. This protocol describes a fast and reliable method for the preparation of barcoded ("indexed") sequencing libraries for Illumina's Genome Analyzer platform. The protocol avoids expensive commercial library preparation kits and can be performed in a 96-well plate setup using multi-channel pipettes, requiring not more than two or three days of lab work. Libraries can be prepared from any type of double-stranded DNA, even if present in subnanogram quantity.

Purification of RNA using TRIzol (TRI reagent).

Abstract: TRIzol solubilization and extraction is a relatively recently developed general method for deproteinizing RNA. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. TRIzol (or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name TRIzol). TRIzol extraction is also an effective method for isolating small RNAs, such as microRNAs, piwi-associated RNAs, or endogeneous, small interfering RNAs. However, TRIzol is expensive and RNA pellets can be difficult to resuspend. Thus, the use of TRIzol is not recommend when regular phenol extraction is practical.

DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements across the Genome from Mammalian Cells

Abstract: Identification of active gene regulatory elements is a key to understanding transcriptional control governing biological processes like cell-type specificity, differentiation, development, proliferation, and response to the environment. Mapping DNase I hypersensitive (HS) sites has historically been a valuable tool for identifying all different types of regulatory elements, including promoters, enhancers, silencers, insulators and locus control regions. This method utilizes DNase I to selectively digest nucleosome-depleted DNA (presumably by transcription factors), whereas DNA regions tightly wrapped in nucleosome and higher order structures are more resistant. The traditional low-throughput method for identifying DNase I HS sites uses Southern blots. Here, we describe the complete and improved protocol for DNase-seq, a high-throughput method that identifies DNase I HS sites across the whole genome by capturing DNase-digested fragments and sequencing them by high-throughput next generation sequencing. In a single experiment, DNase-seq can identify most active regulatory regions from potentially any cell type from any species with a sequenced genome.

Using the metagenomics RAST server (MG-RAST) for analyzing shotgun metagenomes.

Abstract: Shotgun metagenomics creates millions of fragments of short DNA reads, which are meaningless unless analyzed appropriately. The Metagenomics RAST server (MG-RAST) is a web-based, open source system that offers a unique suite of tools for analyzing these data sets. After de-replication and quality control, fragments are mapped against a comprehensive nonredundant database (NR). Phylogenetic and metabolic reconstructions are computed from the set of hits against the NR. The resulting data are made available for browsing, download, and most importantly, comparison against a comprehensive collection of public metagenomes. A submitted metagenome is visible only to the user, unless the user makes it public or shares with other registered users. Public metagenomes are available to all.

Locomotor Activity Level Monitoring Using the Drosophila Activity Monitoring (DAM) System

Abstract: Adult behavioral assays have been used with great success in Drosophila melanogaster to identify circadian rhythm genes. In particular, the locomotor activity assay can identify altered behavior patterns over the course of several days in small populations, or even individual flies. Commercially available, highly efficient automated systems allow for continuous data collection from large numbers of individuals, and analytical tools make it possible to quickly analyze multiple aspects of circadian behavior from each experiment. These features make the locomotor activity assay useful for high-throughput analyses, leading to the rapid discovery and functional characterization of many Drosophila circadian rhythm genes. The locomotor assay described here can simultaneously assess both circadian and sleep behavior, and several methods can be used to analyze the data generated from such assays. This protocol details the use of the Drosophila Activity Monitoring (DAM) System from TriKinetics. Briefly, the system records activity from individual flies maintained in sealed tubes placed in activity monitors. An infrared beam directed through the midpoint of each tube measures an "activity event" each time a fly crosses the beam. Events detected over the course of each consecutive sampling interval are summed and recorded over the course of the experiment for each fly. The general approaches described here can be applied to a wide range of behavioral activity experiments, including sleep deprivation analyses and general studies of hypoactivity and hyperactivity.

In vivo imaging of tumors.

Abstract: INTRODUCTION Light microscopy of tumors, as for other thick, scattering tissues such as the brain or the developing embryo, is limited by light penetration and optical access. because of these problems, epifluorescence and confocal microscopy are typically limited to the outer 50-100 microm of the accessible tumor tissue. Most mouse tumors must be exteriorized for examination under the light microscope, a procedure that limits the duration and repeatability of imaging. this protocol describes the generation of chronic window preparations in the mouse. These preparations allow an implanted tumor to grow for several weeks in an optically accessible location in vivo, making it possible to examine the living tumor with high-resolution light microscopy in a repetitive manner. Two chronic window preparations are described: (1) the dorsal skinfold chamber, which allows in vivo imaging of tumors growing in the subcutaneous space, and (2) the cranial window, which allows in vivo imaging of tumors growing on the brain surface.

Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photoconversion, and FLIP.

Abstract: The technique of fluorescence recovery after photobleaching (FRAP) was introduced in the mid-1970s to study the diffusion of biomolecules in living cells. For several years, it was used mainly by a small number of biophysicists who had developed their own photobleaching systems. Since the mid-1990s, FRAP has gained increasing popularity because of the conjunction of two factors: First, photobleaching techniques are easily implemented on confocal laser-scanning microscopes (CLSMs), and so FRAP has become available to anyone who has access to such equipment. Second, the advent of green fluorescent protein (GFP) has allowed easy fluorescent tagging of proteins and their observation in living cells. Thanks both to the versatility of modern CLSMs, which allow control of laser intensity at any point of the image, and to the development of new fluorescent probes, additional photoperturbation techniques have emerged during the last few years. After the photoperturbation event, one observes and then analyzes how the fluorescence distribution relaxes toward the steady state. Because the photochemical perturbation of suitable fluorophores is essentially irreversible, changes of fluorescence intensity in the perturbed and unperturbed regions are due to the exchange of tagged molecules between those regions. This article first discusses the materials required for performing FRAP experiments on a CLSM and the software for data analysis. It then describes general considerations on how to perform FRAP experiments as well as the necessary controls. Finally, different possible ways to analyze the data are presented.

Variance component methods for analysis of complex phenotypes.

Abstract: Variance component methods have a long history in both human quantitative genetics and agricultural genetics and animal breeding. They are designed for genetic analysis of continuously varying quantitative traits like body mass index (BMI), cholesterol levels, or IQ. They can be used to assess the strength of genetic effects on a trait, to localize genes influencing a trait though either linkage or association methods, to assess whether associated variants are likely to be the functional variants behind a given localization signal, to explore whether related traits have shared genetic influences in multivariate analyses, and to characterize the genetic effects on a trait through analyses of gene-gene and gene-environment interaction. An excellent reference for a thorough explanation of classical variance component methods in genetics is Falconer and Mackay 1996. Conceptually, the idea behind variance component methods is very simple – to decompose the overall variance in a phenotype into particular sources. Assuming that the trait of interest is normally distributed, a common assumption in variance component analyses, the distribution of a trait or phenotype can be described in terms of the mean and variance of the trait. Figure 1 shows the distribution of height in the 1411 participants of the San Antonio Family Heart Study (SAFHS) (Mitchell et al. 1996). The height of study participants ranges from 132.4 cm to 190.5 cm and the mean is 161.64 cm. Most people are about average and a few people are very short or very tall. The variance describes the spread of the trait values around the mean. The variance in height in the SAFHS is 85.65. Asking what the sources of variance in a trait are is essentially asking what makes people different from each other. Figure 1 Distribution of height in the San Antonio Family Heart Study. The most basic way to group these sources of variance is to divide the overall phenotypic variance (σ2p) into genetic (σ2g) and environmental (σ2e) components: σ2p=σ2g+σ2e. (1) Each of these can be further subdivided. Genetic variance is often subdivided into additive and dominance variance and sometimes epistatic variance, which arises from interactions among genes. Environmental variance is typically divided into shared and unshared or unique. Shared environmental variance may reflect influences that are common to members of a nuclear family, to spouses, to sibships, or to larger community units that extend beyond the nuclear family. Unshared or unique environmental variance is specific to each individual and may include things such as measurement error.

β-Galactosidase Assay

Abstract: When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. Although the Escherichia coli lacZ gene, encoding beta-galactosidase (beta-gal), can be used as a standard reporter for monitoring the strength of a promoter or enhancer in a transient or stable transfection assay, it is predominantly used as an internal control during transient transfection experiments. When used in this manner, cells are usually transfected with the control plasmid (containing a ubiquitously active viral promoter fused to the E. coli lacZ gene) and an experimental plasmid containing another reporter gene (e.g., luciferase or chloramphenicol acetyltransferase [CAT]) under the control of the promoter or enhancer of interest. The basic colorimetric assay described here is the simplest and least expensive assay for quantifying beta-gal activity. The cells are lysed and, after determining the total protein concentration in the extracts, an aliquot of the extract is mixed with the reaction substrate, O-nitrophenyl-beta-D-galactopyranoside (ONPG), in a buffer containing sodium phosphate and magnesium chloride. When the yellow product becomes visible, the optical densities of the samples are determined spectrophotometrically.

Culturing and Egg Collection of Aedes aegypti

Abstract: Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.

Processing sleep data created with the Drosophila Activity Monitoring (DAM) System.

Abstract: Adult behavioral assays have been used with great success in Drosophila melanogaster to identify circadian rhythm genes. In particular, the locomotor activity assay can identify altered behavior patterns over the course of several days in small populations, or even individual flies. Sleep is a highly conserved behavior that is required for optimal performance and, in many cases, life of an organism. Drosophila demonstrate a behavioral state that shows traits consistent with sleep: periods of relative behavioral immobility that coincide with an increased arousal threshold after ~5 min of inactivity, regulated by circadian and homeostatic mechanisms. However, because flies do not produce brain waves recordable by electroencephalography, sleep researchers use behavior-based paradigms to infer when a fly is asleep, as opposed to awake but immobile. Data on Drosophila activity can be collected using an automated monitoring system to provide insight into sleep duration, consolidation, and latency, as well as sleep deprivation and rebound. This protocol details the use of Counting Macro, an Excel-based program, to process data created with the Drosophila Activity Monitoring (DAM) System from TriKinetics for sleep analyses. Specifically, it details the steps necessary to convert the raw data created by the DAM System into sleep duration and consolidation data, broken down into the light (L), dark (D), light:dark cycling (LD), and constant darkness (DD) phases of a behavior experiment.

Introduction of Green Fluorescent Protein (GFP) into Hippocampal Neurons through Viral Infection

Abstract: Expression of green fluorescent protein (GFP), its more fluorescent mutant forms (e.g., EGFP [enhanced GFP]), or their fusion protein derivatives, affords a number of informative possibilities in cellular neuroscience. EGFP is a soluble protein and appears to be homogeneously distributed within the cytosol of neurons when expressed. Thus, it reveals the structure of the neuron, including the cell body, and axonal and dendritic arbors. It is also sufficiently bright to reveal detailed structures such as axonal boutons and dendritic spines. When expressed as a fusion protein, EGFP can provide information about the distribution characteristics of the proteins within neurons. Furthermore, during single-cell electrophysiological studies, such expression can direct the investigator to record from a cell carrying a foreign gene. In this protocol, we describe the use of the Sindbis pseudovirus expression system to deliver GFP to neurons. Sindbis is a member of the alphaviruses, which are plus-stranded RNA viruses. This protocol uses the DH(26S) strain, which preferentially infects neurons over glia (50:1). Two infection methods are given: one for dissociated hippocampal cultured neurons and one for organotypic hippocampal slices.

Aedes aegypti: an emerging model for vector mosquito development.

Abstract: Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

Imaging Single Molecules Using Total Internal Reflection Fluorescence Microscopy (TIRFM)

Abstract: Total internal reflection fluorescence microscopy (TIRFM) allows fluorescent molecules to be visualized with an unparalleled signal-to-noise ratio. This is achieved by illuminating only the molecules that are within a thin volume near the coverslip surface but not those that are deeper in solution. Using this technique, fluorescent molecules within approximately 100 nm of the coverslip can be visualized, and single molecules that are separated by a distance greater than the diffraction limit (approximately 200 nm) can be individually resolved. The application of centroid-tracking methods allows subdiffraction-limited localization precision as low as 1 nm. Additionally, by combining centroid-tracking methods with recent advances in fluorophore technology and imaging methods, even those molecules that are present at high concentrations and closer to one another than the diffraction limit can be individually imaged. TIRF is ideally suited for studying protein dynamics on or near the plasma membrane. Although TIRFM was pioneered in the 1980s, it was not until the mid-1990s that single biological molecules were imaged directly. The explosion of new fluorescent proteins, new organic dyes, and quantum dots (Qdots), along with commercially available TIRFMs, has made this technique increasingly useful and accessible to biologists. In this review, we first describe the theory of TIRFM. We then give a detailed description of important considerations for setting up a TIRFM, based on commercially available systems, and review considerations for purification and labeling of proteins. Finally, we discuss new techniques that allow single molecules to be imaged at cellular concentrations and with super-resolution localization.

Preparation of Trojan Horse Liposomes (THLs) for Gene Transfer across the Blood-Brain Barrier

Abstract: Nonviral plasmid DNA is delivered to the brain via a transvascular route across the blood-brain barrier (BBB) following intravenous administration of DNA encapsulated within Trojan horse liposomes (THLs), also called PEGylated immunoliposomes (PILs). The liposome surface is covered with several thousand strands of polymer (e.g., polyethylene glycol [PEG]), and the tips of 1%-2% of the polymer strands are conjugated with a targeting monoclonal antibody that acts as a molecular Trojan horse (MTH). The MTH binds to a receptor (e.g., for transferrin or insulin) on the BBB and brain cell membrane, triggering receptor-mediated transcytosis of the THL across the BBB in vivo, and receptor-mediated endocytosis into brain cells beyond the BBB. The persistence of transgene expression in the brain is inversely related to the rate of degradation of the episomal plasmid DNA. THL technology enables an exogenous gene to be widely expressed in the majority of cells in adult brain (or other organs) within 1 d of a single intravenous administration. Applications of the THLs include tissue-specific gene expression with tissue-specific promoters, complete normalization of striatal tyrosine hydroxylase in experimental Parkinson's disease following intravenous tyrosine hydroxylase gene therapy, a 100% increase in survival time of mice with brain tumors following weekly intravenous antisense gene therapy using THLs, and a 90% increase in survival time with weekly intravenous RNA interference (RNAi) gene therapy in mice with intracranial brain tumors. This protocol describes the preparation of THLs for use in gene transfer in vitro or in vivo.

Stabilization of proteins for storage.

Abstract: Following isolation and purification, it is often necessary to store proteins and peptides for extended periods of time before performing detailed biophysical, enzymatic, and structural proteomics. Therefore, it is essential that the pure target protein maintain its original biological (or functional) behavior over an extended period of storage which may range from weeks to years. Protein pharmaceuticals must remain viable following extensive shipping and storage, and they must remain devoid of all possible inactivation processes. The shelf life of a protein depends on both the intrinsic nature of the protein and the storage conditions. Proteins (especially enzymes) must be stored at an appropriate temperature and pH range and frequently in the presence of concentrated (approximately 1 M) glycerol, sucrose, or a similar substance, for the proteins to retain activity and prevent aggregation. This article discusses the major causes of protein inactivation and describes a range of measures that can be adopted to maintain the stability and solubility of proteins.

Disruption of cultured cells by nitrogen cavitation.

Abstract: Cell disruption by nitrogen decompression from a pressurized vessel is a rapid and effective way to homogenize cells and tissues, to release intact organelles, and to prepare cell membranes. Cells are placed in a pressure vessel and large quantities of oxygen-free nitrogen are dissolved in the cells under high pressure (~5500 kilopascals [kPa], equivalent to 800 pounds per square inch [psi]). When the pressure is released suddenly, the nitrogen bubbles out of solution, rupturing the cell membrane and releasing the cell contents. Nitrogen cavitation is well suited for mammalian and plant cells and fragile bacteria, but is less effective with yeast, fungi, spores, or other cell types with tough cell walls. The chemical and physical stresses imposed by nitrogen cavitation on enzymes and subcellular compartments are minimized compared with ultrasonic and mechanical homogenizing methods. Unlike lysis methods relying on shear stresses and friction, there is no heat damage to proteins and organelles during nitrogen cavitation. Indeed, the method is accompanied by an adiabatic expansion that cools the sample instead. Also, labile cell components are protected from oxidation by the inert nitrogen gas. Furthermore, nitrogen does not alter the pH of the suspending medium. The process is fast and uniform because the same disruptive forces are applied within each cell and throughout the sample, ensuring reproducible cell-free homogenates. Finally, variable sample sizes (e.g., from ~1 mL to 1 L or more) can be accommodated with most commercial systems.

Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB.

Abstract: A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood.

Labeling Cytoskeletal F-Actin with Rhodamine Phalloidin or Fluorescein Phalloidin for Imaging

Abstract: The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The cytoskeleton is composed of a series of filamentous structures, including intermediate filaments, actin filaments, and microtubules. Immunofluorescent staining has been most frequently used to study cytoskeletal components. However, it is also possible to fluorescently label isolated cytoskeletal proteins and either microinject them back into the cell or add them to fixed, permeabilized cells. Alternatively, it is possible to use the mushroom-derived fluorescinated toxins, phalloidin or phallacidin, to label F-actin of the cytoskeleton, as is described in this article. Phalloidin is available labeled with different fluorophores. The choice of the specific fluorophore should depend on whether phalloidin labeling for actin is part of a double-label experiment. In most cells, the abundance of actin filaments should provide a very strong signal. In double-label experiments, the fluorophore should be chosen to take this into account. In general, rhodamine labels are more resistant to photobleaching and can be subjected to the longer exposures required for finer structures.

Digital scanned laser light sheet fluorescence microscopy.

Abstract: Modern applications in the life sciences are frequently based on in vivo imaging of biological specimens, a domain for which light microscopy approaches are typically best suited. Often, quantitative information must be obtained from large multicellular organisms on the cellular or even subcellular level and with a good temporal resolution. However, this usually requires a combination of conflicting features: high imaging speed, low photobleaching, and low phototoxicity in the specimen, good three-dimensional (3D) resolution, an excellent signal-to-noise ratio, and multiple-view imaging capability. The latter feature refers to the capability of recording a specimen along multiple directions, which is crucial for the imaging of large specimens with strong light-scattering or light-absorbing tissue properties. An imaging technique that fulfills these requirements is essential for many key applications: For example, studying fast cellular processes over long periods of time, imaging entire embryos throughout development, or reconstructing the formation of morphological defects in mutants. Here, we discuss digital scanned laser light sheet fluorescence microscopy (DSLM) as a novel tool for quantitative in vivo imaging in the post-genomic era and show how this emerging technique relates to the currently most widely applied 3D microscopy techniques in biology: confocal fluorescence microscopy and two-photon microscopy.

Array Tomography: High-Resolution Three-Dimensional Immunofluorescence

Abstract: Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Although the fabrication procedures can be relatively difficult, the high resolution, depth invariance, and molecular discrimination offered by array tomography justify the effort involved. Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture.

Fluorescence In Situ Hybridization for the Identification of Environmental Microbes

Abstract: This protocol presents methods for the phylogenetic identification of microorganisms in environmental samples (e.g., water and sediments) by means of fluorescence in situ hybridization (FISH) with rRNAtargeted oligonucleotide probes, followed by signal amplification with catalyzed reporter deposition (CARD). In this procedure, FISH probes are conjugated with the enzyme horseradish peroxidase (HRP). After hybridization, the subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities on target cells than after FISH with probes labeled directly with fluorochromes. This protocol describes the custom labeling of tyramides with different fluorochromes, as well as sample preparation and cell permeabilization strategies for various microbial cell wall types. A protocol for sequential multicolor CARD-FISH for the simultaneous detection of different phylogenetic groups is also presented.

Purification of RNA by SDS Solubilization and Phenol Extraction

Abstract: This protocol describes a method for RNA purification by sodium dodecyl sulfate (SDS) solubilization and phenol extraction. It is of wide utility and is used routinely to deproteinize RNAs in biological material that has been solubilized in SDS, an ionic detergent that dissolves membranes, disrupts protein-nucleic acid interactions, and inactivates ribonucleases. Once solubilized, addition of phenol or phenol:chloroform:isoamyl alcohol (PCA) completely denatures the protein, and it becomes insoluble in aqueous solution. PCA extraction is the method of choice for preparing cytoplasmic RNA from tissue culture cells or in any other situation (e.g., enzyme reactions) where solubilization in SDS is easily achievable.

Processing circadian data collected from the Drosophila Activity Monitoring (DAM) System.

Abstract: Adult behavioral assays have been used with great success in Drosophila melanogaster to identify circadian rhythm genes. In particular, the locomotor activity assay can identify altered behavior patterns over the course of several days in small populations, or even individual flies. Generally, circadian behavior is assayed during a period of 12 h light:12 h dark cycling (LD entrainment) followed by conditions of constant darkness (DD). LD activity profiles provide a qualitative image of daily activity bouts, and the data can be used to quantitatively assess the phase and/or amplitude of particular bouts. Additional activity assessments made from entrained flies that have been shifted to constant darkness can provide insight into the state of internal clocks and the ability of these clocks to drive rhythmic outputs. Typical LD DD runs assess both free-running rhythmicity and period length with χ2 periodogram (P'gram) analysis. This protocol describes the use of ClockLab (a MATLAB-based program) and the Counting Macro (an Excel-based program) to analyze circadian locomotor activity data collected using the Drosophila Activity Monitoring (DAM) System from TriKinetics. Specific procedures are described to analyze free-running rhythmicity and period length for individual flies, and assess group activity plots during both entrainment and constant conditions.

Determination of gross chromosomal rearrangement rates.

Abstract: Cells devote a significant amount of metabolism to maintaining the stability of their genome and to preventing inappropriate chromosomal rearrangements that are characteristic of many cancers. A simple genetic assay using haploid derivatives of the yeast Saccharomyces cerevisiae provides a means to quantitatively measure the rate at which gross chromosomal rearrangements (GCRs) accumulate in different genetic backgrounds. This assay measures the rate of simultaneous inactivation of CAN1 and URA3 markers placed on a nonessential end of a yeast chromosome and in principle can be implemented in any haploid strain. Rearrangements detected with this assay include broken chromosomes healed by de novo telomere additions and a spectrum of inter- and intrachromosomal fusion events. The GCR assay allows for detailed analysis of the contributions of individual genes and different pathways in the suppression of genomic instability.

Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells

Abstract: It often is desirable to "prefractionate" RNA before analysis. Ordinarily, this can only be done with tissue culture cells, although it is possible to isolate nuclei and cytoplasm from certain "soft" tissues such as liver and white blood cells. This protocol describes a method for separating nuclei from the cytoplasm that can be used for many tissue culture types. This procedure also is useful for cells grown in suspension or for adherent cells. The procedure relies on swelling in hypotonic buffer, subsequent gentle homogenization, and centrifugation. This method is not appropriate for material (e.g., bacteria, yeast) that has high intrinsic RNase activity, or tissues that are difficult to solubilize, such as muscle tissue or plant material.

Polyacrylamide gel electrophoresis of RNA.

Abstract: Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to or =600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.

Electrophysiological Recordings from the Drosophila Giant Fiber System (GFS)

Abstract: INTRODUCTION: The giant fiber system (GFS) of Drosophila is a well-characterized neuronal circuit that mediates the escape response in the fly. It is one of the few adult neural circuits from which electrophysiological recordings can be made routinely. This protocol describes a simple procedure for stimulating the giant fiber neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS: the tergotrochanteral "jump" muscle (TTM) and the large indirect flight muscles (dorsal longitudinal muscles, or DLMs). It is a relatively noninvasive method that allows the investigator to stimulate the giant fibers in the brain and assay the function of several central synapses within this neural circuit by recording from the thoracic musculature.

Meta-Analysis of Genome-Wide Association Studies

Abstract: Individual genome-wide association studies have only limited power to find novel loci underlying complex traits and common diseases. With relatively modest sample and effect sizes, a true association between genotype and phenotype may never meet genome-wide statistical significance (P < 5 x 10(-8)) in a single study. Through meta-analysis, novel susceptibility loci can be discovered by effectively summing the statistical evidence of individually underpowered studies. Most genetic discoveries for complex traits are now made through meta-analysis collaborations, which so far have been restricted to single-locus analyses, testing for main effects at a single polymorphism at a time. A key benefit of this approach is that individual-level genotype (and phenotype) data do not need to be exchanged between research groups. In this article, we focus on meta-analysis at individual single-nucleotide polymorphisms (SNPs), paying particular attention to how imputation uncertainty can be incorporated into the association analysis and subsequent meta-analysis. Probably the most important aspect of genome-wide association meta-analysis is harmonization of the study results. As studies differ in design, sample collection, genotyping platforms, and association analysis methods, it is important that the association results (per SNP) of each study can be formatted, exchanged, and analyzed in such a way that the statistical evidence can be combined appropriately and that no valuable information is lost. Without minimizing the importance of having a clear phenotype definition (and corresponding measurements), we will assume that investigators representing the various studies have made sensible agreements about phenotype definitions, necessary sample exclusions, and appropriate covariate modeling.