Garc¡a-Ca¤as / Cifuentes / Sim¢ Applications of Advanced Omics Technologies: From Genes to Metabolites

1. Auflage 2014
ISBN: 978-0-444-62669-1
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 64, 496 Seiten

Reihe: Comprehensive Analytical Chemistry

The book contains contributions concerning the application of the new instrumental and methodological developments in omics technologies, including those related to Genomics, Transcriptomics, Proteomics, Peptidomics and Metabolomics, Lipidomics and Foodomics. The16 chapters discuss in detail: innovative applications of functional gene microarrays for profiling microbial communities, microRNA profiling, novel genotyping applications using microarray technology in cancer research, next-generation sequencing applied to the study of human microbiome, emerging RNA-SEQ applications in food science, recent progress in plant proteomics, applications of gel-free proteomic approaches, the challenges and applications of proteomics tools for food authenticity, the role of salivary peptidomics in clinical applications, metabolomic approaches to the study of degenerative, cardiovascular and renal diseases, and neonatal medicine. Also covered are other omics applications such as profiling of genetically modified organisms, the fundamentals, applications and challenges of foodomics, and MS-based lipidomics. Moreover, this volume includes relevant and updated aspects on bioinformatics, data treatment, data integration and systems biology. This book complements the previous volume 'Fundamentals of Advanced Omics Technologies: New Advances from Genes to Metabolites' that covered the fundamental aspects of these new omics technologies. - Describes the latest applications of omics technologies - Provides an excellent reference for applications of advanced omics techniques - Includes advanced tools and methodologies for dealing with the data generated

Autoren/Hrsg.

Garc¡a-Ca¤as, Virginia

Cifuentes, Alejandro

Sim¢, Carolina

Leseproben

Chapter 2 microRNA Profiling
An Overview of Current Technologies and Applications
Sinead M. Smith*; David W. Murray†    * Department of Clinical Medicine and School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland
† Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland Abstract
microRNAs (miRNAs) are small, evolutionarily conserved, noncoding RNAs that posttranscriptionally regulate specific gene products resulting in altered protein expression. miRNAs target specific messenger RNA molecules either by guiding degradation through a mechanism similar to RNA interference or by inhibiting translation. As such, miRNAs control the expression of thousands of genes in a broad spectrum of normal physiological contexts and in disease settings. Recent advances in high-throughput methods for profiling microRNA expression and for the identification of microRNA targets have ushered in a new era in the research of transcriptional regulation. Understanding microRNA expression patterns and microRNA targets provides an insight into gene regulation, biomarker identification, and potential strategies for therapy. This chapter provides an overview of recently developed technologies for the investigation of microRNA expression and function, including profiling by quantitative PCR, microarray analysis, and next-generation RNA sequencing, as well as useful bioinformatics tools for differential expression analysis and target prediction. In addition, the contribution of these miRNA technologies to advances in cancer research is discussed. Keywords microRNA Messenger RNA Expression profiling RNA sequencing Microarray Quantitative PCR 1 Introduction
Recent insight into the transcriptional landscape of mammalian genomes gained from high-throughput next-generation sequencing (NGS) technologies has revealed that although 80% of the genome is transcribed, less than 2% is subsequently translated into protein, resulting in the generation of a large number of ncRNA transcripts (1–5). Although originally considered to be nonfunctional, recent data have highlighted that noncoding RNAs (ncRNAs) play key roles in gene regulation by influencing transcription and translation of target genes, thus regulating a diverse range of biological processes under normal physiological conditions and in disease settings (6). Broadly speaking, ncRNAs may be subdivided according to their size. First, long ncRNAs (lncRNAs) ranging in size from a few hundred nucleotides (nt) to multiple kilobases in length represent the largest class of ncRNAs and account for much of the transcribed genome (6). In contrast to lncRNAs, small ncRNAs are less than 300 nt in length and include microRNAs (miRNAs), piwi-interacting RNAs, small-interfering RNAs, and small nuclear RNAs (snRNAs) among others (7). The miRNAs (18–25 nt) are the most widely studied family of the small ncRNAs. Since their discovery in 1993 (8,9), the diversity and significance of this class of regulatory molecule has become increasingly appreciated. miRNAs posttranscriptionally decrease the expression of thousands of target genes by binding to specific messenger RNA (mRNA) targets and promoting their degradation and/or inhibiting their translation (10–12). Although a relatively limited number of miRNAs (approximately 1000) have been identified in humans compared with the number of mRNAs and proteins (approximately 30,000), a single miRNA may regulate hundreds of mRNAs and thus has the potential to greatly impact gene-expression networks (10). The importance of miRNAs in biological processes is demonstrated by their high levels of evolutionary conservation (13). Accumulating evidence supports a role for miRNAs in normal cellular physiology, where they act as key regulators of development (14), differentiation (15,16), cell proliferation, and apoptosis (17,18). For example, both specific miRNAs and proteins involved in miRNA processing play fundamental roles in the development and function of B- and T-cells within the immune system (19–23). Additionally, miRNAs coordinate cellular responses in innate immune cells (24) and play key roles in the regulation of host–pathogen interactions during infection (25). miRNA expression is also regulated during inflammation (25–27) and as a consequence perturbed miRNA expression is associated with a number of autoimmune diseases, including multiple sclerosis (28), rheumatoid arthritis (29,30), and systemic lupus erythematosus (31,32). Furthermore, expression profiling has implicated miRNAs in numerous cancers, including B-cell chronic lymphocytic leukemia (33) and cancer of the breast (34–37), colon (35,38,39), liver (40,41), and lung (35,42–47), among others. miRNAs function as both tumor suppressors and oncogenes (48,49). Given the key roles of miRNAs in normal cellular homeostasis and their dysregulation under disease conditions, investigation of miRNA expression profiles and regulation of their mRNA targets is essential for a complete understanding of the cell signaling mechanisms that mediate cell function in health and disease, with the potential to identify new therapeutic agents or drug targets. Moreover, as miRNAs are stable in a variety of clinical specimens, including formalin-fixed paraffin-embedded (FFPE) tissue, blood, and urine, there is substantial interest in their development as biomarkers for diagnostic applications. Indeed, profiling of circulating miRNAs has already shown promise for detection of cancers (36,44,45,50–53). 2 miRNA Biogenesis and Nomenclature
An understanding of the multistep processes involved in miRNA processing and biogenesis is required for the design and application of analytical techniques for miRNA detection and quantitation. Primary miRNA (pri-miRNA) transcripts are either transcribed by RNA polymerase II from independent genes or represent introns of protein-coding genes (54–56). A pri-miRNA transcript can contain a single miRNA or multiple miRNAs that are processed from the same transcript (25). The pri-miRNA folds into a hairpin structure, which acts as a substrate for cleavage by the endonuclease Drosha resulting in an approximately 70–100 nt long precursor miRNA (pre-miRNA) (Figure 1). Following the Drosha cleavage, the pre-miRNA is exported to the cytoplasm by Exportin, where it is further processed by the endonuclease Dicer to produce an miRNA duplex comprised of miRNA strands derived from the 5' and 3' regions of the precursor duplex (7,10,55). One strand of this duplex, representing a mature miRNA, is incorporated into the RNA-induced silencing complex (RISC), while the other passenger strand is usually degraded. As part of the RISC, miRNAs base pair with complete or partial complementarity to sequences in the 3' untranslated region (3'-UTR) of target mRNAs and induce mRNA translational repression or instability by deadenylation and degradation (55). Figure 1 miRNA Biogenesis. In the nucleus, pri-miRNA transcripts are either transcribed by RNA polymerase II from independent genes or represent introns of protein-coding genes. Drosha mediates the processing of a pri-miRNA to a pre-miRNA. Following Drosha cleavage, the pre-miRNA is transported to the cytoplasm. Once in the cytoplasm, Dicer processes the pre-miRNA to produce a miRNA duplex derived from the 5' and 3' regions of the precursor. Alternatively, the two strands of the miRNA duplex may be distinguished by the nomenclature miRNA and miRNA*, which indicate the major and the minor strands respectively. Mature miRNAs are loaded into the RISC complex to target specific mRNA molecules for degradation or inhibition of translation. Different mature miRNA species can be produced from a single pre-miRNA molecule, as distinct miRNAs are generated from the 3' and 5' arms of the pre-miRNA duplex. In addition, a given mature miRNA may comprise a distribution of sizes centered around 22 nt rather than a discrete length. The variation in mature miRNA length is due to 3' or 5' end...