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• Isolation of ubiquitylated proteins using tandem ubiquitin-binding entities

  Authors: Aillet F, Lopitz-Otsoa F, Hjerpe R, Torres-Ramos M, Lang V, Rodríguez MS

  Methods in molecular biology (Clifton, N.J.).

  Studying postubiquitylation events has always been a difficult task due to the labile nature of these posttranslational modifications. When utilized in tandem, ubiquitin-binding entities (TUBEs) notStudying postubiquitylation events has always been a difficult task due to the labile nature of these posttranslational modifications. When utilized in tandem, ubiquitin-binding entities (TUBEs) not only increase up to thousand times the affinity for poly-ubiquitin chains but also protect ubiquitylated proteins from the action of the proteasome and de-ubiquitylating enzymes.

• Following protein-glycosaminoglycan polysaccharide interactions with differential scanning fluorimetry

  Authors: K. A. Uniewicz, A. Ori, T. R. Rudd, M. Guerrini, M. C. Wilkinson, D. G. Fernig, E. A. Yates

  Methods in molecular biology (Clifton, N.J.). 836:171.

• The application of microfluidics in biology.

  Authors: David Holmes, Shady Gawad

  Methods in molecular biology (Clifton, N.J.). 583:55-80.

  Recent advances in the bio- and nanotechnologies have led to the development of novel microsystems for bio-particle separation and analysis. Microsystems are already revolutionising the way we doRecent advances in the bio- and nanotechnologies have led to the development of novel microsystems for bio-particle separation and analysis. Microsystems are already revolutionising the way we do science and have led to the development of a number of ultrasensitive bioanalytical devices capable of analysing complex biological samples. These devices have application in a number of diverse areas such as pollution monitoring, clinical diagnostics, drug discovery and biohazard detection. In this chapter we give an overview of the physical principles governing the behaviour of fluids and particles at the micron scale, which are relevant to the operation of microfluidic devices. We briefly discuss some of the fabrication technologies used in the production of microfluidic systems and then present a number of examples of devices and applications relevant to the biological and life sciences.

• Chemical synthesis in microreactors.

  Authors: Paul Watts, Stephen J Haswell

  Methods in molecular biology (Clifton, N.J.). 583:109-20.

  To develop a new generation of drugs, pharmaceutical companies need to be able to synthesise and screen novel chemicals with enhanced speed. New technology that would enable a cost neutral stepTo develop a new generation of drugs, pharmaceutical companies need to be able to synthesise and screen novel chemicals with enhanced speed. New technology that would enable a cost neutral step change in the number of potential drug candidates would provide a distinct competitive advantage. Indeed the miniaturisation of chemical reactors offers many fundamental and practical advantages of relevance to the pharmaceutical industry, who are constantly searching for controllable, information-rich, high-throughput, environmentally friendly methods of producing products with a high degree of chemical selectivity. This chapter reviews the current and future applications of microreactors that could enhance the drug discovery process.

• Microengineered neural probes for in vivo recording.

  Authors: Karla D Bustamante Valles

  Methods in molecular biology (Clifton, N.J.). 583:135-48.

  One of the great challenges facing medicine is the repair of the damaged nervous system. Due to the limited capacity of the central (and to a lesser extent the peripheral) nervous systems toOne of the great challenges facing medicine is the repair of the damaged nervous system. Due to the limited capacity of the central (and to a lesser extent the peripheral) nervous systems to regenerate, damage such as spinal cord injury can often result in permanent paralysis. Researchers are attempting to overcome nerve injury by devising methods of sensing neural activity either in the brain or in the spinal cord or peripheral nervous system. This information can act as a control mechanism for either muscle stimulators (e.g. for restoring limb function) or providing function in some other way (such as controlling a cursor on a computer screen). Ideally, sensing devices are implanted into the body, directly accessing the nervous system. Whilst great advancements have been made in implantable neural stimulators, sensing of neural activity has proven to be a more difficult task. This chapter describes how microengineered probes allow construction of neuron-sized neural interfaces for enhanced recording in vivo.

• Dielectrophoresis as a cell characterisation tool.

  Authors: Kai F Hoettges

  Methods in molecular biology (Clifton, N.J.). 583:183-98.

  Dielectrophoresis (DEP) is a technique which offers label-free measurement of cell electrophysiology by monitoring its movement in non-uniform electric fields. In this chapter, the theory underlyingDielectrophoresis (DEP) is a technique which offers label-free measurement of cell electrophysiology by monitoring its movement in non-uniform electric fields. In this chapter, the theory underlying DEP is explored, as are the implications of the development of equipment for taking such measurements. Practical considerations such as the selection of a suspending medium are also discussed.

• Wireless endoscopy: technology and design.

  Authors: David R S Cumming, Paul A Hammond, Lei Wang

  Methods in molecular biology (Clifton, N.J.). 583:221-46.

  In this chapter we review the current capsule technology and the more conventional "gold standard" technologies against which the wireless devices are compared. Over the years there have been severalIn this chapter we review the current capsule technology and the more conventional "gold standard" technologies against which the wireless devices are compared. Over the years there have been several implementations of capsule devices of growing sophistication as new technology has become available. A notable feature is the extent to which the devices available at any given time have relied upon other more mainstream technologies from which capsule builders have been able to borrow. As an inevitable consequence, device complexity and functionality have increased.

• Prediction of Plant miRNA Genes.

  Authors: Matthew W Jones-Rhoades

  Methods in molecular biology (Clifton, N.J.). 592:19-30.

  This chapter presents procedures for the computational identification of plant miRNA genes. In the first procedure, homologs of known miRNAs are identified in a database of genomic or cDNA sequence.This chapter presents procedures for the computational identification of plant miRNA genes. In the first procedure, homologs of known miRNAs are identified in a database of genomic or cDNA sequence. In the second procedure, previously unidentified miRNA families are predicted through the analysis of secondary structure, evolutionary conservation, and targeting potential.

• miRNA Target Prediction in Plants.

  Authors: Noah Fahlgren, James C Carrington

  Methods in molecular biology (Clifton, N.J.). 592:51-7.

  In plants, miRNA bind to target RNAs with a high degree of complementarity. In this chapter, a simple method for computationally predicting plant miRNA targets, using a position-dependent scoringIn plants, miRNA bind to target RNAs with a high degree of complementarity. In this chapter, a simple method for computationally predicting plant miRNA targets, using a position-dependent scoring system, is described.

• Directed Gene Silencing with Artificial MicroRNAs.

  Authors: Rebecca Schwab, Stephan Ossowski, Norman Warthmann, Detlef Weigel

  Methods in molecular biology (Clifton, N.J.). 592:71-88.

  The characterization of gene function typically includes a detailed analysis of loss-of-function alleles. In model plants, such as Arabidopsis thaliana and rice, sequence-indexed insertionThe characterization of gene function typically includes a detailed analysis of loss-of-function alleles. In model plants, such as Arabidopsis thaliana and rice, sequence-indexed insertion collections provide a large resource of potential null alleles that can often be easily accessed through convenient Web sites (e.g., http://signal.salk.edu ). They are, however, not available for nonmodel species, require stacking for knockout of redundant homologs, and do not easily allow for partial or regulated loss of gene function, which is particularly useful when null alleles are lethal. Transgene approaches that employ directed gene silencing can substitute for null alleles and also enable refined studies of gene function, e.g., by tissue-specific and inducible gene-silencing.This chapter describes the generation and application of artificial microRNAs (amiRNAs) as a gene silencing tool in a wide variety of different plant species.

• High-Throughput Approaches for miRNA Expression Analysis.

  Authors: Cheng Lu, Frédéric Souret

  Methods in molecular biology (Clifton, N.J.). 592:107-25.

  miRNAs have emerged as key regulators of gene expression in both plants and animals. These small (generally 21-22 nt) RNA molecules, originated from primary "hairpin" transcripts, can inducemiRNAs have emerged as key regulators of gene expression in both plants and animals. These small (generally 21-22 nt) RNA molecules, originated from primary "hairpin" transcripts, can induce translational suppression or direct mRNA cleavage. Similar to regular mRNAs, the expression of miRNAs is highly regulated. Their expression pattern could provide critical clues to understanding miRNA functions. However, many previously identified miRNA families have multiple paralogous loci. Within each family, different members are often closely related and sometimes give rise to identical miRNAs. This poses critical challenges in the analysis of individual miRNA genes. This chapter describes several methods that are commonly used for miRNA expression analysis, including high-throughput sequencing, microarrays, and briefly discusses qRT-PCR, northern blotting, and other approaches used for data validation.

• Analysis of miRNA Modifications.

  Authors: Bin Yu, Xuemei Chen

  Methods in molecular biology (Clifton, N.J.). 592:137-48.

  After transcription, a large number of cellular RNAs employ modifications to increase their diversity and functional potential. Modifications can occur on the base, ribose, or both, and are importantAfter transcription, a large number of cellular RNAs employ modifications to increase their diversity and functional potential. Modifications can occur on the base, ribose, or both, and are important steps in the maturation of many RNAs. Our lab recently showed that plant microRNAs (miRNAs) possess a 2'-O-methyl group on the ribose of the 3' terminal nucleotide, and that this methyl group is added after miRNA/miRNA* formation. One function of this modification is to protect miRNAs from 3' terminal uridylation by an unknown enzymatic activity. It is possible that uridylation of miRNAs triggers their degradation. Here we describe a protocol to purify a specific miRNA in order to determine its molecular mass so that the presence of a modification can be inferred, an in vivo method to detect 3' terminal modification of miRNAs, and an (alpha-(32)P) dATP incorporation assay to study 3' terminal uridylation of miRNAs.

• Computational Methods for Comparative Analysis of Plant Small RNAs.

  Authors: Gayathri Mahalingam, Blake C Meyers

  Methods in molecular biology (Clifton, N.J.). 592:163-81.

  Small RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specificSmall RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specific target genes and loci. Most if not all plants utilize these small RNA signaling networks. We have developed a deep-sequencing based dataset of plant small RNAs, based on the hypothesis that comparisons among the complex pool of small RNAs from diverse plants will identify novel types of conserved, regulated, or species-specific molecules. A database containing upward of hundreds of millions of plant small RNA sequences is being created for comparative analyses. This small RNA database will allow the experimental characterization of the majority of the biologically important small RNAs for a range of plant species. This database can be accessed from our website ( http://smallrna.udel.edu/ ). A variety of web-based tools have been developed for analyses of these data. Here, we focus on these tools, and we describe how the users can implement these tools to analyze and interpret the small RNA data and how the users could use similar approaches for other sets of plant small RNAs from diverse species.

• Abiotic Stress-Associated miRNAs: Detection and Functional Analysis.

  Authors: Dong-Hoon Jeong, Marcelo A German, Linda A Rymarquis, Shawn R Thatcher, Pamela J Green

  Methods in molecular biology (Clifton, N.J.). 592:203-30.

  MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at theMicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.

• Purification of Arabidopsis Argonaute Complexes and Associated Small RNAs.

  Authors: Yijun Qi, Shijun Mi

  Methods in molecular biology (Clifton, N.J.). 592:243-54.

  Argonaute (AGO) proteins recruit small RNAs to form effector complexes of RNA interference (RNAi), collectively termed RNA-induced silencing complexes (RISCs). Here, we describe detailed protocolsArgonaute (AGO) proteins recruit small RNAs to form effector complexes of RNA interference (RNAi), collectively termed RNA-induced silencing complexes (RISCs). Here, we describe detailed protocols for the purification of AGO complexes and their associated small RNAs, using Arabidopsis AGO1 as an example.

• Piecing the Puzzle Together: Genetic Requirements for miRNA Biogenesis in Arabidopsis thaliana.

  Authors: Zhixin Xie

  Methods in molecular biology (Clifton, N.J.). 592:1-17.

  MicroRNAs (miRNAs) are an important class of endogenous small silencing RNAs in both plants and animals. They regulate the expression of a wide range of target genes that are involved in manyMicroRNAs (miRNAs) are an important class of endogenous small silencing RNAs in both plants and animals. They regulate the expression of a wide range of target genes that are involved in many important biological processes. Biogenesis of plant miRNAs requires a distinct set of proteins, including members that belong to several highly conserved RNA silencing protein families. The framework for miRNA biogenesis in plants was revealed through genetic and biochemical analyses using mutants that are defective in miRNA accumulation. These general miRNA-deficient mutants constitute a set of invaluable genetic resources for the plant miRNA research community. They could be utilized to experimentally validate the candidate miRNAs that are either predicted by a computational program or recovered from a small RNA deep sequencing effort which is becoming a more affordable and widely used approach for small RNA discovery. Starting with a brief introduction on multiple small RNA pathways in plants, this chapter provides basic experimental procedures for the examination of miRNA accumulation from wild type plants and various mutant lines in Arabidopsis.

• Methods for Isolation of Total RNA to Recover miRNAs and Other Small RNAs from Diverse Species.

  Authors: Monica Accerbi, Skye A Schmidt, Emanuele De Paoli, Sunhee Park, Dong-Hoon Jeong, Pamela J Green

  Methods in molecular biology (Clifton, N.J.). 592:31-50.

  For the experimental analysis of miRNAs and other small RNAs in the 20-25 nucleotide (nt) size range, the first and most important step is the isolation of high-quality total RNA. Because RNAFor the experimental analysis of miRNAs and other small RNAs in the 20-25 nucleotide (nt) size range, the first and most important step is the isolation of high-quality total RNA. Because RNA degradation products can mask or dilute the presence of true miRNAs, it is important when choosing a method that it efficiently extracts RNA from tissues in a manner that prevents degradation of RNA of both high and low molecular weight. In addition, the presence of polyphenols, polysaccharides, and secondary metabolites may render nucleic acids insoluble, and hinder the recovery of the miRNAs. Finally, and most importantly, the method chosen must be capable of retaining the small RNA component. In this chapter, we will present a set of total RNA isolation methods that can be used to maximize the recovery of high-quality RNA to be used in miRNA analysis for a large number of plant species and tissue types.

• A Method to Discover Phased siRNA Loci.

  Authors: Michael J Axtell

  Methods in molecular biology (Clifton, N.J.). 592:59-70.

  Short, interfering RNAs (siRNAs) arise from the processing of long double-stranded RNA (dsRNA) by Dicer enzymes. Dicers generate siRNA duplexes by successive hydrolysis of both strands of the dsRNAShort, interfering RNAs (siRNAs) arise from the processing of long double-stranded RNA (dsRNA) by Dicer enzymes. Dicers generate siRNA duplexes by successive hydrolysis of both strands of the dsRNA phosphodiester backbone at positions determined by measuring 21-24 nucleotides from an exposed dsRNA terminus. Therefore, a population of dsRNAs with precisely identical termini will produce siRNA spaced in regular, 21-24-nucleotide intervals. This chapter presents an easily customized and generally applicable strategy for identifying loci which produce the "phased" siRNAs diagnostic of such processing. Given the input of a large set of expressed small RNAs and of the corresponding genome or transcriptome from which the small RNAs are derived, the methodology produces a ranking of user-defined loci with respect to their likely production of phased siRNAs. Top ranked loci are candidates for further computational and biological analyses.

• Bioinformatics Analysis of Small RNAs in Plants Using Next Generation Sequencing Technologies.

  Authors: Kan Nobuta, Kevin McCormick, Mayumi Nakano, Blake C Meyers

  Methods in molecular biology (Clifton, N.J.). 592:89-106.

  Next-generation sequencing technologies have a substantial impact on a broad range of biological applications. Like many other groups, we use these new technologies, especially SBSNext-generation sequencing technologies have a substantial impact on a broad range of biological applications. Like many other groups, we use these new technologies, especially SBS (Sequence-By-Synthesis), for deep profiling of small RNA molecules in plants. Small RNAs are 21-24 nucleotides in length and are known to play a major role in the activation of mRNAs and genomic DNAs. We have generated numerous SBS small RNA libraries; each can consist of more than three million signatures of more than 33 nucleotides in length. Here, we describe the challenges and our strategies to handle the very large quantity of small RNA data generated by these next-generation sequencing technologies.

• In Situ Detection of miRNAs Using LNA Probes.

  Authors: Zoltán Havelda

  Methods in molecular biology (Clifton, N.J.). 592:127-36.

  A spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of understanding the precise biological functions of miRNAs. Since miRNAs are very short molecules, theirA spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of understanding the precise biological functions of miRNAs. Since miRNAs are very short molecules, their in situ analysis is technically demanding. Here, we describe a protocol for miRNA in situ detection in plants based on LNA-modified oligonucleotides probes. LNA modification significantly enhances the sensitivity and specificity of miRNA detecting probes and provides relatively easy in situ miRNA detection.

• MicroRNA Promoter Analysis.

  Authors: Molly Megraw, Artemis G Hatzigeorgiou

  Methods in molecular biology (Clifton, N.J.). 592:149-61.

  In this chapter, we present a brief overview of current knowledge about the promoters of plant microRNAs (miRNAs), and provide a step-by-step guide for predicting plant miRNA promoter elements usingIn this chapter, we present a brief overview of current knowledge about the promoters of plant microRNAs (miRNAs), and provide a step-by-step guide for predicting plant miRNA promoter elements using known transcription factor binding motifs. The approach to promoter element prediction is based on a carefully constructed collection of Positional Weight Matrices (PWMs) for known transcription factors (TFs) in Arabidopsis. A key concept of the method is to use scoring thresholds for potential binding sites that are appropriate to each individual transcription factor. While the procedure can be applied to search for Transcription Factor Binding Sites (TFBSs) in any pol-II promoter region, it is particularly practical for the case of plant miRNA promoters where upstream sequence regions and binding sites are not readily available in existing databases. The majority of the material described in this chapter is available for download at http://microrna.gr .

• Biotic Stress-Associated microRNAs: Identification, Detection, Regulation, and Functional Analysis.

  Authors: Florence Jay, Jean-Pierre Renou, Olivier Voinnet, Lionel Navarro

  Methods in molecular biology (Clifton, N.J.). 592:183-202.

  The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant inThe methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcripts that were repressed in wild-type seedlings treated with a biotic stress, (2) a 5' RACE-derived assay to map miRNA target sites, and (3) a bioinformatic analysis to retrieve specific miRNA loci from the Arabidopsis genome. We then describe methods used to monitor (1) the levels of primary miRNA transcripts (pri-miRNAs)/mature miRNAs and (2) the transcriptional activity of miRNAs in response to a biotic stress and bacterial challenge. Furthermore, we present a strategy to identify additional biotic stress-responsive miRNA genes and get insight into their regulation. This involves (1) a microarray approach that allows detection of pri-miRNAs, coupled with (2) a promoter analysis of co-regulated miRNA genes. Finally, we describe strategies that can be used to functionally characterize individual biotic stress-associated miRNAs, or the miRNA pathway, in disease resistance.

• Processing of miRNA Precursors.

  Authors: Yukio Kurihara, Yuichiro Watanabe

  Methods in molecular biology (Clifton, N.J.). 592:231-41.

  Plant microRNA (miRNA) processing requires at least two cleavage steps of respective precursors. The first cleavage step is from pri-miRNA to pre-miRNA, and the second cleavage step is from pre-miRNAPlant microRNA (miRNA) processing requires at least two cleavage steps of respective precursors. The first cleavage step is from pri-miRNA to pre-miRNA, and the second cleavage step is from pre-miRNA to mature miRNA. Using northern blot analysis, we previously showed that the RNase III enzyme Dicer-like protein 1 (DCL1) and the double-stranded RNA-binding (DRB) protein HYL1 are involved in the processing reactions of miRNA precursors. The processing of plant miRNAs differs from that of animal miRNAs in some respects. Here, we introduce our methods for analyzing the processing of miRNA precursors through transient expression of mutated pri-miRNAs with modified stem-loop structures in Nicotiana benthamiana. The methods described here could be useful for understanding how DCL proteins and cognate DRB proteins are involved in adequate recognition and processing of substrate precursor RNA molecules.

• Transient Assays for the Analysis of miRNA Processing and Function.

  Authors: Felipe F de Felippes, Detlef Weigel

  Methods in molecular biology (Clifton, N.J.). 592:255-64.

  Transient assays provide a convenient alternative to stable transformation. For small RNA analysis in plants, the most widely used method, commonly named agroinfiltration, makes use of AgrobacteriumTransient assays provide a convenient alternative to stable transformation. For small RNA analysis in plants, the most widely used method, commonly named agroinfiltration, makes use of Agrobacterium tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Agroinfiltration has been used successfully in many different applications, including the analysis of small RNAs. We describe here a protocol for analysis of miRNA processing using agroinfiltration of N. benthamiana leaves.

• Microfabrication techniques for biologists: a primer on building micromachines.

  Authors: Douglas Chinn

  Methods in molecular biology (Clifton, N.J.). 583:1-53.

  In this chapter we review the fundamental techniques and processes underlying the fabrication of devices on the micron scale (referred to as "microfabrication"). Principles laid down in the 1950s nowIn this chapter we review the fundamental techniques and processes underlying the fabrication of devices on the micron scale (referred to as "microfabrication"). Principles laid down in the 1950s now form the basis of the semiconductor manufacturing industry; these principles are easily adaptable to the production of devices for biotechnological processing and analysis.

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