Methods in Molecular Biology™ Series Editor John M Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to www.springer.com/series/7651 RNA Interference From Biology to Clinical Applications Edited by Wei-Ping Min Departments of Surgery, Microbiology and Immunology, and Pathology, University of Western Ontario, London, ON, Canada Thomas Ichim MediStem Laboratories Inc., San Diego, CA, USA Editors Wei-Ping Min, MD, Ph.D Departments of Surgery Microbiology and Immunology, and Pathology University of Western Ontario London, ON Canada mweiping@uwo.ca Thomas Ichim, Ph.D MediStem Laboratories Inc San Diego, CA USA thomas.ichim@gmail.com ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-587-3 e-ISBN 978-1-60761-588-0 DOI 10.1007/978-1-60761-588-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010920843 © Springer Science+Business Media, LLC 2010 All rights reserved This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein Printed on acid-free paper Humana Press is a part of Springer Science+Business Media (www.springer.com) Preface There are a few moments, defining the research path of one’s career that remain crystal clear and as memorable as yesterday For both of us, one such moment was our learning of the process of RNA interference and the stunning realization of its implications in our discipline Being immunologists by training, we have been interested in exploring how to either activate this T cell more toward one direction or manipulate this dendritic cell in another We have been used to doing this through different tissue culture conditions, or addition of chemical inhibitors: these having the drawbacks of unscalability and unspecificity, respectively It was a cold Canadian night in the winter of 2001 We were having coffee at the Hospital Cafeteria waiting for some data coming out of the laboratory, and both of us were talking about the future of immunology The need for specific ways of modulating genes so that we would be spared of the need for impractical approaches was discussed “How exciting would it be just to use antisense oligonucleotides to silence immune stimulatory genes in the dendritic cell?” “It must have been performed already.” “If it has, then why don’t we know about it?” “It’s much easier to evoke a therapeutic effect by modulating immunological genes, in that, unlike viral or oncogenes, even a 20% gene inhibition will cause a biological response.” “Someone must have done that with antisense already.” As this was before the time everyone had a Blackberry and an iPhone, we would have to wait until we got upstairs to check Pubmed But we continued the conversation: “Is there anything better than antisense? What about ribozymes?” And this led to the discussion regarding RNA interference At that time, the concept of RNA interference was still restricted mainly to the world of molecular biologists We remembered a dear friend telling us about this bizarre phenomena, whereby introduction of a double strand of RNA would induce cleavage not only of the introduced nucleic acids but also any other nucleic acids that resembled it He told us about this being the “next antisense” since it is part of the body’s endogenous defenses against viruses and therefore theoretically should be more potent for silencing “It’s easier to take away a gene than add one.” “Yes, but the double strands would activate interferon responses – The paper our friend told us about was in worms.” “But imagine if there was a way to get around that? Plus if you use it to suppress immune suppressive cytokines in cancer then the interferon alpha response is actually beneficial.” We left our coffees and hurriedly went to the computers upstairs to see what has and has not been done in this field We printed out everything that had the words “RNA interference” the 1998 Nature paper that described RNA interference in worms (which subsequently won Fire and Mello the Nobel Prize), the paper by Elbashir et al showing that the interferon alpha response can be avoided in mammals, the work describing use of siRNA for studying mammalian genes That night, neither of us had much sleep thinking about the possibility of specifically silencing immunological genes We had a perfect model, the dendritic cells, which reside at the center of the “immunological universe,” and are relatively simple cells to transfect and manipulate, Wei-Ping having already induced them to express various immune regulatory genes such as FasL v vi Preface Initial silencing of the interleukin-12 p35 gene was performed The degree of knockdown was phenomenal These data led us into a journey that continues today, having silenced both immune suppressive and immune stimulatory genes ranging from cytokines, to membrane proteins, to oncogenes, to transcription factors This journey has taken us personally from ex vivo cell manipulation to current cell-targeting immunoliposomes that deliver siRNA to dendritic cells only, thus alleviating the need for hydrodynamic injection Disease models treated have included rheumatoid arthritis, allergy, transplant rejection, and cancer When we were contacted by Humana with the possibility of being editors for this volume, we gladly accepted it In the same way that we described our personal journey, we aimed in this book to represent the journey of our field From those early days where RNA interference was a strange artifact in worms, to the 2006 Noble Prize to Fire and Mello, to the current clinical trials and the $1 billion purchase of a siRNA company by Merck, the field of RNA interference has grown at a breakneck pace In this volume, we will overview the science and the Protocols at present that span the biological disciplines from detailed nucleic acid chemistry, to pharmacology, to manipulation of signal transduction pathways By compiling an overview of the different ongoing areas of scientific investigation of RNAi, we hope to two things: stimulate new questions and provide you with the tools to start addressing those questions The book is divided into three main segments The first deals with the Physiology of RNA Interference, in which we try to overview the biological relevance of this process and provide a context for the next sections The second section, entitled “RNA interference in the laboratory and siRNA delivery” outlines practical uses of RNAi either as research tools or as components in the development of therapeutics Finally, the last part of the book deals with actual preclinical and clinical issues associated with the use of RNAi-inducing agents as drugs Through this clustering of chapters in segments, we hoped to provide a logical context for the current state of the art Starting the first section, Drs Abubaker and Wilkie from University of Guelph, Canada provide a comparative biology examination of the relevance of RNAi processes to viral defense They overview commonalities and differences between gene silencing effector mechanisms and host–parasite interactions in forms of life ranging from fungi, to worms, to insects, to mammals Subsequent to establishing an overall framework for understanding the various biological pathways associated with RNA interference as a gene-specific mechanism of defense, they move into a discussion on innate defense mechanisms, namely the ability of double-stranded RNA molecules to activate the interferon alpha response through activation of toll like receptors (TLR) 7/8 and the acid inducible gene I (RIG-I) In the subsequent chapter, Drs Gantier and Williams from Monash University in Clayton, Australia review the relevance of this “danger-associated” TLR pathway as a method of immune activation and provide methodology for assessment, in both mouse and man, of its activation RNAi-induction by microRNA (miRNA) also plays a role of fundamental innate protection mechanisms against pathogens The miRNA can be pre-existing in the host cell or can be transcribed by the invading virus Drs Ouellet and Provost from Laval University in Canada, go into considerable detail across the major viruses to discuss the impact of host and viral miRNA in the battle for survival Of particular interest are the analytical methods for detection of even transiently expressed miRNAs The exquisite sensitivity and selectivity of RNAi induction allows for knock-down of specific alleles of a gene Dr Hohjoh from the National Institute of Neuroscience in Tokyo, Japan, provides protocols for silencing of the Photinus and Renilla luciferase genes Preface vii in mammalian cells The same selectivity that allows for allele-specific silencing by siRNA also requires great care in designing siRNAs, in that numerous factors contribute to silencing efficacy The issue of siRNA-designing algorithms is reviewed by Dr Kim from the University of Science & Technology in Daejeon, Korea who presents the AsiDesigner, a web-based siRNA design program that takes into consideration alternative splicing in designing optimum siRNAs Drs Muhonen and Holthöfer from Dublin City University, Dublin, Ireland, continue on the theme of optimizing siRNA design by discussing issues of target messenger accessibility and provide various bioinformatics approaches for identifying active and specific sites on the mRNA for silencing Dr Ishigaki’s group from the Kanazawa Medical University, Kanazawa, Japan, describes another method of increasing potency of siRNA In their chapter, shRNAs are expressed on a single plasmid, so that by concurrently targeting different areas of the same transcript, increased silencing may be achieved They proved a detailed protocol for generating dual shRNA expressing plasmids and describe various methodological peculiarities of this approach Of particular relevance to therapeutic development, the authors detail possible adverse effects by overconsumption of cellular transcription machinery when various promoters of shRNA transcription are used Practical application of multi-shRNA derived from a single plasmid could include suppression of HIV Drs Rossi and Zhang from the Beckman Research Institute, City of Hope, CA, address this possible therapeutic approach through disclosing their technique involving a new combinatorial anti-HIV gene expression system that allows for simultaneous expression of multiple RNAi effector units from a single Pol II polycistronic transcript In their system, they avoid the cell toxicity associated with expressing numerous shRNAs from Pol III promoters by using endogenous RNAi transcripts and miRNAs for expression of multiple RNAi effector units off a single Pol II polycistronic transcript University of Vienna’s Dr Hofacker, subsequently discusses in silico tools that consider only siRNA-specific design criteria and those that integrate mRNA structure features as well as basic siRNA features for selection of shRNA and siRNAs The final chapter of the First Section is by Dr Engels et al from J.W Goethe-Universität in which protocols for synthesis of various siRNAs are provided In the Second Section, we transition from the biology of RNA interference to issues related to implementation, both in the laboratory setting as a basic research reagent and as a potent tool useful for the development of therapeutics for diseases Dr Zheng et al from University of Western Ontario, Canada, begin the section by describing methodology for producing cell-targeting siRNA-bearing immunoliposomes Through the ability of immunoliposomes to selectively bind to antigen-expressing cells corresponding to the antibody on the immunoliposome, the investigators provide a delivery platform that is relatively simple to generate and has widespread applications The original method of in vivo siRNA delivery, hydrodynamic injection, is reviewed in the next chapter by Drs Evers and Rychahou from the University of Texas This method involves a rapid administration of high volume siRNA intravenously, which temporarily causes micropores and loosening of tight junctions in the endothelium, causing siRNA entry across the plasma membrane into intracellular compartments To date, this method has been used to deliver siRNA to the liver, lungs, and brain In the same way that DNA array technologies have allowed for en masse identification of gene expression patterns in various cells and biological conditions, the knock-down of genes using high throughput siRNA technologies has allowed for the understanding of cellular phenotypes after a gene is suppressed Fujita et al from the Research Institute for Cell Engineering (RICE) and the National Institute of Advanced Industrial Science and viii Preface Technology (AIST), Tokyo, Japan, describe two protocols for reserve transfection of siRNA molecules on solid surfaces, the first for microarrays and the second for microtiter plates Moving from general to specific, the use of siRNA in specific pathologies is examined in greater detail Prakash et al from McGill University, Canada, are focused on neurodegenerative diseases and the means of traversing the blood brain barrier They provide a detailed review of the state of the art regarding neurological uses of siRNA and subsequently describe the generation of optimized siRNA sequences and delivery methods for in vivo targeting using cationic nanoparticles Huang et al from the Chang Gung Memorial Hospital-Kaohsiung Medical Center in Taiwan used a bioinformatics approach to selectively identify genes in lung cancer through random knock-down and assessment of phenotype Using this approach, they identified FLJ10540, a target associated with cancer invasion and migration In their chapter, they describe upstream and downstream control of this tumor-associated factor Delivery of siRNA and shRNA, of particular interest to cancer models, is described in the Chapter of Drs Jere and Cho (Seoul National University, Korea) who provide protocols for generation of biodegradable cationic polymers Methods of tracking cellular update and intracellular trafficking as well as protocols for the evaluation of the impact on cancer cells are provided While selective delivery of RNAi-inducing molecules has been performed with immunoliposomes or affinity-targeting agents, an interesting approach is described by Ohtsuki’s group from Okayama University in Okayama, Japan, who used HIV-tat conjugation of siRNA to allow intracellular delivery and could activate the gene silencing process using photons This novel method, termed CPP-linked RBP-mediated RNA internalization and photoinduced RNAi (CLIP-RNAi), could have many applications in therapeutic scenarios where localized silencing is desirable The issue of siRNA degradation is examined by Aigner et al who utilize various polyethylenimines to increase protection from nucleases, both extracellular and intracellular In their chapter, the authors provide a comparison of the different polyethylenimines in respect to cationic charge, ability to form noncovalent interactions with siRNA, and compaction of the siRNA into complexes that allow for internalization by endocytosis On the same topic of crossing the plasma membrane, Brito et al from King’s College, London, England provide a rather interesting transfection methodology: temporary permeabilization with streptolysin-O They provide protocols that have been optimized for gene silencing of multiple myeloma cell lines, which have great importance for therapeutics development The third section of the book covers the issue of clinical implementation of RNAi A look at www.clinicaltrials.gov , the NIH registry for ongoing clinical trials, reveals seven ongoing clinical investigations using RNAi induction for conditions such as wet macular degeneration, infectious diseases, and cancer The current chapter will address some of the issues that need to be addressed in the translation of this new class of therapeutic approaches Dr Akaneya from the Osaka University Graduate School of Medicine, Japan, begins by describing the advantages and disadvantages of using RNAi-inducing approaches for neurological conditions Specific diseases discussed include ALS and inflammatory conditions Issues such as immunogenicity, interferon response, and localization are discussed Drs Mao and Wu from Johns Hopkins School of Medicine, Baltimore, describe specifics of using RNAi-based approaches in cancer immunotherapy They discuss various important immunological targets starting with specific effector molecules, and then moving on to more general upstream transcription factors such as STATs and other global regulators of numerous immune response genes The issue of endogenous miRNA controlling of the immune response, both natural and stimulated, is also overviewed Preface ix The authors conclude by evaluating various RNAi-inducing approaches for the most rapid clinical translation in immunotherapy of cancer Tissue injury prevention by RNAi strategies is discussed by Zhang et al from University of Western Ontario, Canada They provide details of assays used to assess renal injury in an ischemia/reperfusion model and prevention by suppression of caspase transcription From the same Institute, Drs Zhang and Li present protocols for the in vitro silencing of dendritic cells with siRNA and subsequent use of these cells to modulate and/or suppress transplant rejection The advantage of this approach is the potent immune stimulatory/ immune suppressive ability of DC dependent on expression of costimulatory molecules Targeting of RelB, an NF-kB family member, is demonstrated in the protocols, which causes suppression of various cytokine and costimulatory molecules on the dendritic cell, this suppression associated with inhibited immunogenicity Continuing on the theme of immune modulation, Ritprajak et al from Tokyo Medical and Dental University, Tokyo, Japan utilize siRNA to enter across the stratum corneum and into dermal dendritic cells By modulating these cells, the authors describe suppression of costimulatory molecules and possible use for treatment of allergic disease Sarret et al from University of Sherbrooke, Canada, use RNAi to tackle the problem of pain in a nonpharmacological manner They discuss protocols for siRNA administration, targets, and behavioral systems used in researching this unique approach to pain management, with particular reference to G protein-coupled receptors Seth et al from MDRNA Inc, Bothell, USA, describe the use of RNAi in treatment of respiratory viruses, with emphasis on influenza They describe various viral targets, animal models, and methods of delivery for maximum antiviral activity An interesting subject is the interaction between siRNA that stimulates interferon alpha responses and the overall antiviral activity of these molecules Drs Malek and Tchernitsa from the Institute of Pathology, Charité – Universitätsmedizin Berlin, Germany and Oncology Institute of Southern Switzerland provide detailed protocols for silencing of ovarian cancer cells in vitro and in vivo Of particular interest is the clinically relevant human xenograft ascites model that is described As you may see, the progress of RNA interference research has been significant The question of whether it will deliver on its promise is still open; however, we hope this volume will provide to you, our reader, the same amount of excitement we’ve had in seeing the field progress to where it is today 2009 Wei-Ping Min and Thomas Ichim Contents Preface v Contributors xiii Part I Physiology of RNA Interference Endogenous Antiviral Mechanisms of RNA Interference: A Comparative Biology Perspective Abubaker M.E Sidahmed and Bruce Wilkie Monitoring Innate Immune Recruitment by siRNAs in Mammalian Cells Michael P Gantier and Bryan R.G Williams Current Knowledge of MicroRNAs and Noncoding RNAs in Virus-Infected Cells Dominique L Ouellet and Patrick Provost Allele-Specific Silencing by RNA Interference Hirohiko Hohjoh Computational siRNA Design Considering Alternative Splicing Young J Kim Bioinformatic Approaches to siRNA Selection and Optimization Pirkko Muhonen and Harry Holthofer Optimized Gene Silencing by Co-expression of Multiple shRNAs in a Single Vector Yasuhito Ishigaki, Akihiro Nagao, and Tsukasa Matsunaga Strategies in Designing Multigene Expression Units to Downregulate HIV-1 Jane Zhang and John J Rossi Designing Optimal siRNA Based on Target Site Accessibility Ivo L Hofacker and Hakim Tafer 10 Chemical Synthesis of 2′-O-Alkylated siRNAs Joachim W Engels, Dalibor Odadzic, Romualdas Smicius, and Jens Haas 21 35 67 81 93 109 123 137 155 Part II RNA Interference in the Laboratory and siRNA Delivery 11 siRNA Specific Delivery System for Targeting Dendritic Cells Xiufen Zheng, Costin Vladau, Aminah Shunner, and Wei-Ping Min 12 Hydrodynamic Delivery Protocols Piotr G Rychahou and B Mark Evers 13 New Methods for Reverse Transfection with siRNA from a Solid Surface Satoshi Fujita, Kota Takano, Eiji Ota, Takuma Sano, Tomohiro Yoshikawa, Masato Miyake, and Jun Miyake 14 Nonviral siRNA Delivery for Gene Silencing in Neurodegenerative Diseases Satya Prakash, Meenakshi Malhotra, and Venkatesh Rengaswamy xi 173 189 197 211 436 Malek and Tchernitsa Welsh, J.B., Zarrinkar, P.P., Sapinoso, L.M., Kern, S.G., Behling, C.A., Monk, B.J., et al (2001) Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer Proc Natl Acad Sci U.S.A 98, 1176–1181 Adib, T.R., Henderson, S., Perrett, C., Hewitt, D., Bourmpoulia, D., Ledermann, J., and Boshoff, C (2004) Predicting biomarkers for ovarian cancer using gene-expression microarrays Br J Cancer 90, 686–692 Rogalla, P., Drechsler, K., Frey, G., Hennig, Y., Helmke, B., Bonk, U., and Bullerdiek, J (1996) HMGI-C expression patterns in human tissues: Implications for the genesis of frequent mesenchymal tumors Am J Pathol 149, 775–779 10 Gattas, G.J., Quade, B.J., Nowak, R.A., and Morton, C.C (1999) HMGIC expression in human adult and fetal tissues and in uterine leiomyomata Genes Chromosomes Cancer 25, 316–322 11 Rogalla, P., Drechsler, K., Kazmierczak, B., Rippe, V., Bonk, U., and Bullerdiek, J (1997) Expression of HMGI-C, a member of the high mobility group protein family, in a subset of breast cancers: relationship to histologic grade Mol Carcinog 19, 153–156 12 Meyer, B., Loeschke, S., Schultze, A., Weigel, T., Sandkamp, M., Goldmann, T., et al (2007) HMGA2 overexpression in non-small cell lung cancer Mol Carcinog 46, 503–511 13 Abe, N., Watanabe, T., Suzuki, Y., Matsumoto, N., Masaki, T., Mori, T., et al (2003) An increased high-mobility group A2 expression level is associated with malignant phenotype in pancreatic exocrine tissue Br J Cancer 89, 2104–2109 14 Chau, K.Y., Manfioletti, G., Cheung-Chau, K.W., Fusco, A., Dhomen, N., Sowden, J.C., et al (2003) Derepression of HMGA2 gene expression in retinoblastoma is associated with cell proliferation Mol Med 9, 154–165 15 Miyazawa, J., Mitoro, A., Kawashiri, S., Chada, K.K., and Imai, K (2004) Expression of mesenchyme-specific gene HMGA2 in 16 17 18 19 20 21 22 23 24 squamous cell carcinomas of the oral cavity Cancer Res 64, 2024–2029 Andrieux, J., Demory, J.L., Dupriez, B., Quief, S., Plantier, I., Roumier, C et al (2004) Dysregulation and overexpression of HMGA2 in myelofibrosis with myeloid metaplasia Genes Chromosomes Cancer 39, 82–87 Berlingieri, M.T., Manfioletti, G., Santoro, M., Bandiera, A., Visconti, R., Giancotti, V., and Fusco, A (1995) Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells Mol Cell Biol 15, 1545–1553 Pentimalli, F., Dentice, M., Fedele, M., Pierantoni, G.M., Cito, L., Pallante, P., et al (2003) Suppression of HMGA2 protein synthesis could be a tool for the therapy of well differentiated liposarcomas overexpressing HMGA2 Cancer Res 63, 7423–7427 Malek, A., Bakhidze, E., Noske, A., Sers, C., Aigner, A., Schafer, R., and Tchernitsa, O (2008) HMGA2 gene is a promising target for ovarian cancer silencing therapy Int J Cancer 123, 348–356 Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate EMBO J 20, 6877–6888 Yuan, B., Latek, R., Hossbach, M., Tuschl, T., and Lewitter, F (2004) siRNA Selection Server: an automated siRNA oligonucleotide prediction server Nucleic Acids Res 32(Web Server issue), W130–W134 Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference Nucleic Acids Res 32, 936–948 Amarzguioui, M and Prydz, H (2004) An algorithm for selection of functional siRNA sequences Biochem Biophys Res Commun 316, 1050–1058 Khvorova, A., Reynolds, A., and Jayasena, S.D (2003) Functional siRNAs and miRNAs exhibit strand bias Cell 115, 209–216 Index A A20 329, 336 A450 276, 279 A600 275 ABI PRISM 7700 system 200, 204 Acepromazine 216, 220 Acetonitrile 157, 158, 161–163 ACK Lysing buffer 359 Active AKT 234, 240–241 Adeno-associated viruses (AAVs) 320, 321 Adenoviruses 46, 51–53, 55, 56, 320 Agarose gel .74, 112, 115–117, 184, 251, 252, 294, 344, 354, 410, 413, 428 Agarose NEEO 286 Akt 244, 247, 255–262 AKT-Ser473 phosphorylation 236 Albino Alexa Fluor 546 C5 maleimide .273, 275, 277, 278 Alexa Fluor 546 fluorophore (TatU1A-Alexa) 272 Alexa546-labeled TatU1A (TatU1A-Alexa) 273–276, 278 Aliphatic amines 249 Alkylated phosphoramidites 159 Allele-specific RNAi (ASP-RNAi) .67, 71, 77 Alphaherpesvirus Varicella 51 Alternative splicing 81–91, 96 A549 lung epithelial cell line 399, 403 Amyotrophic lateral sclerosis (ALS) 214, 320, 321 Anchorage dependent 260 Anhydrous acetonitrile 157 Anhydrous dichloromethane (aDCM) 246, 249, 250, 265, 266 Anion-exchange chromatography 164 Annexin V-FITC 248, 259–260 Annexin-V-Fluos 427 Annexin-V staining 431 Anthocyanin Anti-ERK2 antibodies 301 Anti-GAPDH antibodies 301, 307 Antigenomic pode 56 Antigen-presenting cells (APCs) 328, 357, 373 Antiproliferative 43, 50 Antisense oligonucleotides (AO) 138, 155, 174, 316, 342 Apex 391, 395 Apoptosis 10, 28, 38, 47, 48, 50, 56, 200, 257, 259–260, 268, 318, 330, 332, 342, 344, 352–353, 424, 431 Arabidopsis thaliana 13, 14 Arginine-glycine-aspartic acid 220 Argonaute (Ago2) 7, 11, 36–38, 155 Arthropod-borne viruses 10 Astrocytes 318 Athymic nude mice .286, 294, 432 Atropine 402, 414 Autoradiography 39, 125, 130, 294 Autosomal recessive disorder 114 Avanti Polar Lipids (Alabaster, AL) 175 B B2 11, 13 BACH-1 50 Bak 332 BALB/c mice .175, 186, 369, 377, 400, 402, 418 BamHI 44, 343, 344, 346 Bam HI-A region rightward transcript (BART) 44–48 Bam HI fragment H rightward open readingframe (BHRF1) 44, 48 Basic Local Alignment Search Tool (BLAST) 89, 102, 103, 149, 212, 225, 434 Bax 332 Bcl-2 49, 332 BCLX (BCL2L1) gene 88–91 Bcl-xL (NM_138578) 89–91 Bcl-xS (NM_001191) 89–91 Beckman Coulter DU800 spectrophotometer 176, 178 Beta-actin .113, 118, 235, 237, 411, 425, 426, 428, 430, 433 Betadine antiseptic solution 191, 193 Betaherpesvirus 48 Bicinchoninic acid 300 Bijoux bottles 248 Biocompatibility 284, 287, 292 Biodegradable 215, 225, 243–268 Biodistribution 190, 286–287, 292–294 Biogenesis 14, 36–38, 40, 53, 54, 93 Bioinformatic analyses 101 Biomarkers 39 437 RNA Interference 438 Index Bio-Rad Econo chromatography column 176, 180 Bipolar electrodes 318 Bisaminophosphine 161 Bis-(N,N-diisopropylamino)-2cyanoethoxyphosphine (P2) .157, 160–162 BK virus (BKV) 51, 52 Blood-neural barrier (BNB) 317, 320 Blood-retinal barrier 318 Blotting buffer 426 Blunt-end siRNAs 22, 27 BMDC 181–183, 185 Bombyx mori .11 Boranophosphate modification 316 Bovine serum albumin (BSA) .182, 232, 274, 276, 300–302, 399, 400, 402, 405, 408, 409, 414, 418 Branched 10 kD PEI F25-LMW 284, 287 Bright Line-Haemocytometer 300 Brilliant SYBR Green QPCR Master Mix 177, 360, 364 Bromophenol blue 112, 125, 128, 233, 300, 343 Budker 189 Buprenorphine 193 Burkitt’s lymphoma (BL) 44, 45 C C3 342, 344, 346–349, 351, 353 C5a 344, 346–347 Caenorhabditis elegans 5, 6, 9, 11–13, 314 Calcein-AM 200, 205–208 Cary Eclipse mass spectrofluorometer 176, 180 Caspase 342–344, 346–349, 351, 353 Caspase 200, 205, 206, 344, 346–347, 353 Castleman’s disease 49 Cationic nanoparticle 219–220 Cationic polymers 216, 219, 220, 226, 267, 435 CCL17 331, 336 CCL22 331, 336 CD4+ .58, 326, 328–332 CD8+ 48, 326, 328, 330–332 CD40 46, 177, 181–183, 185, 357, 360, 364 CD80 .182, 358, 365, 374 CD86 182, 357, 358, 360, 364, 365, 374–377 CD11C 357 CD86 costimulatory molecule 374 CD1 mice 342 cDNA 14, 23, 29, 31, 113, 177, 184, 291, 347–349, 360, 363, 409–413 CD40 siRNA immunoliposomes (CD40 siILs) 185 Cell microarrays 198 Cell-penetrating peptides (CPPs) 272, 274 CEM cells .124, 127, 131, 133 Centri-Sep 273, 275 Certificate of Analysis (COA) 417 Chalcone synthase Chemiluminescence 225, 301 Chemokines 48, 331 Chinese hamster ovary (CHO) .276, 277, 279, 280 Chitosan 216, 219, 220, 408 Chlorhexidine solution 191, 193 Chloroform 178, 179, 183, 187, 217, 223, 290, 347, 363, 412 Chloro-N,N-diisopropylamino-2cyanoethoxyphosphine (P1) 161 Chloro phosphine 161 Choi’s algorithm 82 Chromatin remodeling 55 Chromatography 110, 125, 157, 158, 161, 162, 164, 166, 176, 180, 234, 273, 275, 284, 286, 287, 293, 294, 301 ChsA Cisterna magna 391 CL1-0 A549, 232 CL-4B 176, 178, 180 Complement 49, 342 Complement-binding protein 49 Complete Dulbecco’s modified Eagle’s medium (DMEM) 22, 28, 29, 69, 112, 116, 124, 198, 200–202, 206–208, 285, 346, 347, 375, 376, 399, 400, 402, 404–406, 416, 425 Complexation 284, 287–289, 292, 295, 317 Complexes 26, 36, 97, 116, 221, 226, 238, 251, 252, 254, 257, 267, 284–289, 291–294, 296, 315, 317, 335, 344, 362, 416 Confocal laser scanning microscopy (CLSM) 247, 257 Coomassie Brilliant Blue (CBB) 277 Copper grids 247, 253 Co-suppression 4, 12 Co-target mRNA 84, 88 CPP-conjugated 272 c-Rel 344, 358 CT26 colon cancer cells 334 CTLA-4 333, 334, 373, 374 Cucumber mosaic virus strain (CMV-Y ) 14 Cy-3 194 Cy-5 194 Cyclin D 49 Cyclodextrin polycations 216 Cy3-labeled 175, 179, 183, 185, 199, 203, 204, 208 Cytokine assay 419–420 Cytomegalovirus (CMV) 8–10, 40, 48–49, 247 Cytometer 254, 255, 300, 303, 369, 370, 419 Cytometry 177, 181, 182, 185, 247, 255, 260, 302, 364, 365, 376, 377 Cytopathic effects (CPE) 405, 407 RNA Interference 439 Index Cytotoxicity 200, 203–204, 215, 216, 219, 267, 284, 287, 289 Cytotoxic T lymphocyte-associated antigen (CTLA-4) 333, 334, 373, 374 Cytotoxic T lymphocytes (CTL) 52 D 700 Da 246 2000 Dalton polyethylene glycol (PEG2000) 175, 179 Decapentaplegic homologue (SMAD-3) 10, 333 DEDDh nuclease family 13 Deleterious genes 313 Dendrimers 216, 219, 271 Dendritic cells (DCs) .24, 173–187, 327–332, 335–336 Dengue virus 10 Deoxy-nucleotide triphosphate (dNTP) 23, 29, 177, 184, 285, 291, 360, 410, 412, 413 Desilylation 158 Detritylation 158, 164 Dextran 199, 202, 203, 208, 400 Dialysis membrane 246, 250, 266 1,2-Diaminocyclohexane-N,N,N’,N’tetraacetic acid 286 Dicer 3, 6–8, 10, 13, 14, 36, 37, 53–56, 58, 59, 139, 155, 243, 315, 316, 326, 327, 358, 384, 385 Dicer-like (DCL) enzymes 14 Dicer substrate siRNA (DsiRNA) 384–385, 387–393 Dichloromethane (aDCM)(CH2Cl2) 157, 158, 161, 162, 169, 246, 249, 265 4,5-Dicyanoimidazole (DCI) 157, 161, 162 Diethylether 164 Diethylpyrocarbonate (DEPC)-H2O 158, 163, 165, 177, 183, 217, 222, 286, 291, 347, 359, 363, 400, 410, 412 Difco 112, 248 Diffuse large B-cell lymphomas (DLBCL) 48 DiGeorge syndrome critical region (DGCR8) 36, 37 DIgR2 330, 336 Dihydrofolate reductase 49 Dimethoxytrityl (DMTr) 160, 162, 164 4,4’-Dimethoxytrityl (DMTr) group 160 Dimethyldioctadecylammonium bromide (DDAB) 175 Dimethyl sulfoxide (DMSO) 200, 232, 273, 431 2,4-Dinitro-1-fluorobenzene (DNFB) 375, 377, 379 Distearoylphosphatidylethanolamine-PEG2000 (DSPE-PEG2000) 175, 178 Dithiothreitol (DTT) 23, 177, 233, 300, 360 DL-Dithiothreitol 300 DMTr-On oligo 165 DNA polymerase 49, 111, 113, 115–117, 401, 413 Dopaminergic neurons 320 Dorsal root ganglia (DRG) 391, 392 DOTAP 23–26, 31, 408 DOTMA 408 Double stranded RNA (dsRNA) 3, 28, 93, 139–140, 155, 214, 243, 271, 314, 326–328, 358, 415 DPBS 367, 400, 407 DraIII site .111, 115, 116 Driving pulse 318–319 Drosha 7, 36–37, 54, 58, 315, 326–327 Drosophila 6, 11–13, 36, 94, 319 Drosophila C virus (DCV) 11 Dual-Glo Luciferase Buffer 404 Dual luciferase assay 404 Ducks 37 E EBV See Epstein-Barr virus EBV-associated nuclear antigen (EBNA1) 45 E coli 12, 116, 275, 343, 345, 346, 431, 432 E coli BL21(DE3) 275 EcoRI 115, 127, 427, 431 EDTA 68, 69, 112, 124, 125, 177, 178, 198, 201, 207, 232, 233, 246, 248, 258, 274, 285, 286, 293, 300, 399, 413 EF-TEM 253, 267 EGFP gene (pEGFP) 110, 114–115, 247, 254, 255, 278 Ego-1 12 Electrophoresis 74–75, 111–113, 119, 125, 128, 131, 166, 219, 233–234, 240, 251, 252, 294, 301, 304, 344, 402, 410, 413, 428, 429 Electrophysiology 319 Electroporation 315, 317–320 ELISA 23, 24, 27, 125, 127, 132, 133, 248, 258, 292, 394, 416, 419 Encapsulation 186, 190, 321 Endonuclease 7, 321 Endonucleotic cleavage 315 Endosome 271–272, 276, 284, 328 Energy-filtering transmission electron microscopy (EF-TEM) 253, 267 Energy valley 99, 100 Enflurane 349 Enhanced chemiluminescent (ECL) 234, 241, 242, 301, 306, 307, 426, 429 env gene 54 Episome 44 Epstein-Barr virus (EBV) 8, 9, 44–48, 56 Epstein-Barr virus-encoded small RNAs (EBERs) 56 ERI-1 13 ERK2 siRNA 300, 306–307 RNA Interference 440 Index Ethanol (EtOH) 29, 73, 75, 78, 112, 116, 128, 158, 163, 177, 183, 199, 200, 202, 217, 222, 223, 240, 247, 256, 290, 347, 352, 359, 361–363, 365, 400, 409, 412, 413, 427 Ethidium bromide (EtBr) 74, 112, 113, 117, 184, 246, 251, 253, 267, 413 Ethidium homodimer-1 (Eth-D1) 200, 204 Eukaryotic initiation factor 2a(eIF2a) 53 Euthanasia V solution 415 Exocyclic amino 158, 160, 163 Exportin-5 .7, 36–37, 56, 315 Extinction coefficient 166, 167, 169, 178, 218 Extracellular transfection .251, 314, 317 F Fas 330, 344 Fas ligand (FasL) 330, 336 Fas-ligand inhibitory protein (FLIP) 49, 50 FASTA 88, 89, 96, 144, 147, 148, 150 FastPrep 400, 401, 409 Ferret 398, 402, 413–415, 420, 421 Fetal bovine serum (FBS) 22, 26, 69, 112, 116, 124, 176, 180, 182, 186, 198, 201, 206, 232, 233, 238, 247, 248, 254, 258, 260–262, 274, 300, 302, 303, 343, 346, 347, 359, 360, 362, 375, 376, 399, 400, 404–406, 416 Fetal calf serum (FCS) 285, 288, 425, 429 Fibronectin 199, 202, 207 Ficoll-Hypaque 402, 415 Ficoll-Paque 176, 185, 186, 360, 370 Ficoll-Paque Plus 22, 26 Ficoll-Paque solution 360, 366 Firefly luciferase (Luc) 115, 194, 434 FlexiPlate siRNA 198, 200 FL1-H channel 431 FLJ10540 232–238, 242 Flock house virus (FHV) 11, 13 Flow activated cytometry sorting (FACS) 302 Flow cytometry 177, 182–183, 247, 360, 364–365 Flp-In-CHO cell line 277 Flp-In-recombination system 277 Fluor 546 .272–275, 277, 278 Fluorescein 375, 431 Fluorescence 39, 180, 203–205, 207, 255, 276, 277, 279, 280, 370, 431 Fluorescence microscope 177, 182, 185, 199, 200, 203, 204, 274 Fluorochrome 303, 360, 368, 375–377 F-12 medium 274, 276 Formaldehyde 255, 294, 360, 385 Functional genomics 81, 174, 299, 301 Fungi .3, 4, 13, 285 G b-Galactosidase (lacZ) 68–72, 76–78, 194 Gammaherpesvirus 44, 49 [g-32P]ATP 286, 293 GAPDH 29, 30, 114, 177, 185, 301, 306–307, 348–350, 360, 411 GC content 82, 85, 89, 97–99, 138, 141 Gene delivery 244, 249, 330 Gene knockdown 137, 192, 200, 201, 204–206, 218, 283–296, 326, 334, 424, 430, 432 Gene knockout 358 GenePix 4200A 200, 207 GenePorter 177, 181–182, 185 Gene silencing .3–7, 11, 13–15, 38, 55, 67, 72, 78, 82, 89, 91, 93, 101, 109–121, 123, 137, 174, 175, 185, 211–227, 271, 272, 276, 278, 315, 316, 326–328, 334, 335, 349, 351, 357–370, 423–435 Gene targeting 225, 286, 289, 292, 424, 427, 433 Gene transfection 177, 346–347 Genome 4, 5, 9, 11–13, 40, 44, 48–57, 59, 83, 84, 89, 127, 214, 247, 315, 320, 335, 396, 428 Gentamicin solution 402, 414 Giemsa solution 239 GL2 284 GL3 284, 434 Global translational repression 214 Good laboratory practice (GLP) 248 GPCR See G protein coupled receptors G protein coupled receptors (GPCRs) 49, 387, 388 Granulocyte/Macrophage colony-stimulating factor (GM-CSF) 176, 180, 359, 362 Granzymes 342 Green fluorescent protein (GFP) 110, 194 Guanidinium thiocyanate 285, 290 Guanidinoethyl 157 GW182-containing bodies (GW-bodies) 37 H H1 110 H1299 232, 237 Habituation 388, 389 Hairpin RNA 3, 8, 52, 53, 57, 68, 110, 123, 127, 243, 271, 330, 398, 403, 431 Ham’s F-12 medium 274, 276 HCV See Hepatitis C virus HEK293 124, 131, 236, 242 HeLa cells 59, 89, 116, 120, 198, 201–206, 208, 403 Helicase 7, 8, 315 Hemacytometer 26, 28, 248, 259, 416 Hemagglutination assay 405–407, 409 Hematopoietic cell differentiation 38 Hematopoietic cells 48, 260 Hematoxylin and eosin (H&E) 352–354 RNA Interference 441 Index Hemolysis 394 Heparin 26, 247, 252 Hepatitis 8, 56, 57 Hepatitis B virus (HBV) 57 Hepatitis C virus (HCV) 8–10, 57, 58 Hepatitis delta antigen (HDAg) 57 Hepatitis delta virus (HDV) 56, 57 HEPES-KOH 111, 168–169, 273, 374 Herpes simplex virus-1 (HSV-1) 9, 42–44 Herpes simplex virus thymidine kinase (HSV-TK) 69, 70 Herpesviridae 40–51 Herpesvirus 40, 42, 44, 45, 48–51 Heteroduplexes 156 Hexanucleotides 285 HHV-7 51 Highly active antiretroviral therapy (HAART) 58 High performance liquid chromatography (HPLC) .158, 163–169 Hind III 67 Hippocampus 318, 320 Histidine-lysine (HK) 317 Histone acetyltransferase 59 Histone deacetylase HDAC-1 55 HIV .124, 126, 321 HIV-1 encephalopathy (HIVE) 59 HIV-1 Tat peptide 272 [3H-labeled] thymidine (Amersham) 178, 186 HMGA2 gene 426–433 H-NMR 246, 250, 266 H5N1 pandemic 398 Hodgkin Reed-Sternberg tumors 333 Hodgkin’s lymphoma (HL) 44 Homing mechanism 342 Homologous 4, 7, 52, 412 Homology 3–5, 10, 82, 103, 104, 219, 247 Horseradish peroxidase (HRP) 27, 114, 119, 132, 135, 225, 344, 354, 417, 426 HOSE cell line 428 Host-encoded miRNA 9, 15 Housekeeping gene 29, 291, 348, 350 HPLC buffer 158 HSV-2 See Human herpes virus type Hsv1-miR-H1 42 HT1080 124 1H-tetrazole 161, 162 hTLR7 24, 32 hTLR8 24, 26, 32 Human Cu/Zn superoxide dismutase (SOD1) 320, 321 Human cytomegalovirus (HCMV) 8, 41, 48–49, 56 Human endogenous retrovirus L (HERV-L) 55 Human hepatocellular carcinoma (HHC) 57 Human herpesvirus type (HSV-2) 42–44 Human herpesvirus type (HHV-3) 51 Human herpesvirus type (HHV-4) 44 Human herpesvirus type (HHV-5) 48 Human herpesvirus type (HHV-6) 51 Human herpesvirus type (HHV-8) 49 Human immunodeficiency virus type (HIV-1) 8, 10, 39, 48, 54, 55, 58, 123–135, 272, 320 Human papillomavirus 329, 330, 332 Human papillomavirus type-16 .329, 330, 332 Human PBMCs 24–27, 32 Human retrovirus Human T cell leukemia/lymphoma virus type (HTLV-1) 54, 55 Human T98G cells 22, 28, 30 Human TLR8 .22–25, 27, 31 Huntington’s disease 214, 215, 321 Hybridoma 176, 178 Hydrodynamic 189–194 Hydrolytic 266, 267 2-Hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) 23, 176, 178, 179, 216, 218, 284, 399, 400, 426 Hygromycin B 274, 277 Hyperactivation 328 Hypervolemia 392 Hypochromicity 166 Hypothalamic nuclei 318 I ICP4 43, 44 ICP34.5 43, 44 IE72/IE1 suppression 49 I element 12 IFN-b 9, 58, 140 IFN-g .329–331, 336 IFN-responding pathway 48 IKKe 331 IL-2 419 IL-4 .176, 180, 359 IL-6 49, 329, 332, 419 IL-7 329 IL-10 332, 419 IL-12 329, 332 IL-13 419 IL-15 329 IL-1b 419 IL12 p40 419 IL12 p70 419 2-Iminothiolane (Traut’s reagent) 176 Immature DCs (imDC) 357, 370 Immediate-early protein IE72/IE1 48 Immune modulation 50, 172, 173 Immunogenicity 24, 215 Immunohistochemistry 185, 353–354 RNA Interference 442 Index Immunohistology 318 Immunoliposome 173, 175–176, 179, 180, 186 Immunological complications 215 Immunomodulation 48 Immunorecognition 139 Immunoregulatory 332, 357, 358, 374 Immunoregulatory capacity 374 Immunostimulation .22, 24, 32 Immunostimulatory motifs 102–104 Immunostimulatory RNAs (isRNAs) 102 Immunosuppression 45, 373 Indoleamine-2,3-dioxygenase (IDO) 329, 330, 336 Infected-cell protein (ICP0) 42–44 Infectious mononucleosis 44 Inferior vena 192, 351 Influenza virus 397, 398, 403, 405, 413, 414 Innate immunity 7, 21, 27, 418 In silico .24, 38, 144, 214 In situ .39, 293, 352 Integral membrane protein 2A (ITM2A) 50 Integrase 54 Interferon (IFN) 7–9, 24, 28, 49, 53, 58, 94, 102, 316, 318, 379, 417 Interferon regulatory factor 49 Interferon response 94, 109, 121, 140, 214 Interferon responsive genes (IRG) 214 Interphase 26, 227, 390, 412 Intracerebroventricular Injection (ICVI) 220 Intranasal 408–409, 413–415, 418, 420, 421 Intraperitoneal (i.p.) injection 317, 349, 418, 424 Intraportal .189, 192–194 Intrathecal infusion 387 In vacuo 162 Invasion assay 233, 236, 239 In vivo 5, 15, 24, 32, 37, 44, 55, 56, 155, 174, 175, 185, 189, 190, 194, 211, 214–216, 219, 220, 225, 249, 254, 283–296, 314–320, 329–334, 343, 350, 351, 376, 377, 379, 383–395, 398, 400, 402, 408, 418, 423–435 IPTG 275 IQ5 Multicolor i-cycler 29 IRAK1 331 Ischemia 341, 342, 344, 350–352, 355 Ischemia reperfusion (I/R) injury 341, 342, 355 Iscove’s modified DMEM (IMDM) 285, 402, 415, 416 Isoflurane 192–194, 286, 293, 295, 385, 388 Isoform 82–84, 87–91, 96, 97 Isopropanol .177, 183, 227, 290, 347, 359, 363, 412 Isopropyl b-D-1-thiogalactopyranoside 273 J Jamestown Canyon virus ( JCV) 41, 51, 52, 55 JetPEI 284, 287, 288, 292 Jugular 192 K Kanamycin 198, 201, 206, 273, 275, 427, 431 Kaposin gene (K12) 50 Kaposi sarcoma (KS) 8, 50, 399, 400, 402 Kaposi sarcoma-associated herpes virus (KSHV) 8, 9, 41, 43, 49–51, 58 KCl 23, 216, 234, 273, 285, 301, 401, 413, 426 Ketamine 193, 216, 349, 386, 390, 391, 400, 402, 408, 414, 418 Ketamine/xylazine hydrochloride solution 400 Ketamine/xylazine solution 193 Keyhole limpet hemocyanin (KLH) 173, 185, 186, 316, 366, 368 KH2PO4 23, 216 Kinases 236, 329 Kinetochore KSHV genome 49, 50 L Label IT TrackerTM CX-Rhodamine kit 247, 255 lacZ 189, 194 Latency .9, 10, 42, 44, 45, 50, 55, 58 Latency-associated membrane protein 2a (LMP2A) 45 Latency-associated nuclear antigen (LANA) 50 Latency-associated transcript (LAT) 42–44, 50 Latency I 45 Latency II 45 Latency III 45, 48 Latent infection 40, 45, 47 Latent membrane protein (LMP1) 9, 46 LB medium .73, 273, 275, 346 L929 cell line 176, 182, 183, 342, 343, 346, 349 LDH 296 Lentivirus 50, 54, 320 L-glutamine 343, 400 Ligand 10, 49, 175, 190, 200–202, 330, 336, 374 Light intensity 278 Lipids See Lipoplex LipofectamineTM 233, 238, 247, 254, 255 LipofectamineTM RNAiMAX 2000 .399, 402, 403, 405 Lipofectamine 2000 Transfection Reagent 69, 78, 199, 202, 203 Lipoplex 175, 317, 392 Liposomes 175, 178–183, 185, 190, 220, 315, 317, 321, 408 LMax II Luminometer 399 Local free energy (∆Gloc) 101 Locked Nucleic AcidTM (LNA) 39, 156 Long terminal repeat (LTR) 54, 55 LSR II flow cytometer 300, 303 Luciferase 50, 69–72, 77, 111, 189, 194, 284, 286, 288–290, 295, 399, 403–405, 434 Luciferase assay .286, 290, 295 Luciferase quantitation kit 289 Lumbar spinal cord 391, 392 Luminometer 69, 289, 290, 399, 404 Lymphocyte extravasation 342 Lyophilized siRNA .218, 374, 376 Lysate 29, 72, 76, 94, 118, 120, 183, 224, 236, 275, 290, 303–305, 347, 409, 420 Lysosomes 284 M Machinery 7, 8, 10, 11, 13, 114, 214, 215, 244, 284, 335 Macular degeneration 321 Magnesium sulphate (MgSO4) 157, 273 MAP kinase 232 Matrigel 233, 235, 236, 239, 248, 261, 264 Mature miRNAs 37, 39, 40, 42, 43, 52, 126, 327 MD5 MDCK cells 399, 403, 405, 406 Medium 22, 25, 26, 29, 31, 69, 73, 75, 76, 78, 112, 116–118, 124, 131, 176, 180–182, 185, 186, 198, 201–203, 206, 232, 233, 238, 239, 247, 248, 254–265, 268, 273–277, 285, 288–290, 300, 302, 303, 308, 343, 346, 347, 359, 361, 362, 365–368, 370, 375, 376, 399, 400, 402, 404–407, 415, 416, 425, 429 MEK 232, 236 Melanoma-differentiation-associated gene 2-Mercaptoethanol .113, 176, 180, 182, 295, 343, 359 b-Mercaptoethanol (b-ME) 233, 273, 277, 300, 301, 401 Messenger RNA (mRNA) 35, 36, 43, 51, 94, 96, 314, 347, 424 Metastasis 231, 244, 248, 255, 257, 261 Methanol 114, 119, 161, 165, 167, 200, 203, 205, 234, 239, 240, 263, 301, 306, 400, 426 MgCl2 .68, 216, 273, 285, 401, 410, 412, 413 MHC class II molecules 177, 182, 328, 358 MHC class I molecules 328 MHC-class-1-polypeptide-related sequence B (MICB) 10, 48, 49 Microarray analysis 50, 232 Microarray scanner 200 Microcentrifuge 26, 118, 217, 224, 268 Micro RNAs 6, Microtiter platers 198, 199, 201–204, 207, 208, 366, 367, 399, 416 Millipore Ultra-centrifugal Devices (Millipore) (MWCO) of 100 kDa 175, 178, 180, 250, 266 Minimum infectious dose 50 (MID50) 408, 414 Mini-PROTEAN Electrophoresis System 233 Mini Trans-Blot cell system 301, 305 RNA Interference 443 Index miR-1 9, 58 miR-30 9, 58, 431 miR-32 8–10 miR-128 9, 58, 59 miR-155 50, 58, 331, 336 miR-196 9, 58 miR-296 9, 58 miR-351 9, 58 miR-431 9, 58 miR-448 9, 58 miRBase 36 miRDeep 40 miR-I 44 miR-LAT miR-N367 54 miRNA seed 38 MiRNeasy Easy mini kit 217 miRNP 36, 37, 56, 315 miR-TAR-3p 55 miR-TAR-5p 55 Mirtrons 36 Mitochondrial enzyme complex I 56 mivaRII-138 miRNA 53 MLR 178, 186 mM-EDTA solution (Nacalai Tesque) 198 M-MuLV Reverse Transcriptase 285, 291 Mn: 258 575, 246 MnCl2 (10% v/v) 175, 179 Mn: 423 Da 246 Mock 30, 54, 255, 257, 258, 430 Mock control 31, 434 Modified 2X Laemmli buffer 300, 304 Molecular weight cut off (MWCO) 175, 178, 180, 250, 266 Monkey SV40 virus 51 Monophosphate 22, 28, 32 3’ Monophosphate .22, 28, 30 MOPS (3-(N-morpholino) propanesulfonic acid 286 MOPS buffer 286, 294 Morphology 253, 357 Mortar 217, 222, 290, 293, 294 Mouse cytomegalovirus (mCMV) 40 Mouse TLR7 22–25, 31 Mouse TNF-a (mTNF-a) 24, 27 MTT assay 232–233, 235, 238, 242, 296 Multi-shRNA 126 Murine herpes Murine renal ischemia 344 Mutagenesis 320 Mutant SOD1 (SOD1G93A) 321 MWG 284, 285 Myalgia 413 Mycoplasma 285, 295 Myeloma 299–308 RNA Interference 444 Index Myeloproliferative disorders 428 Myofilament Myriad 174 MySQL relational database (RDBMS) 85, 89 N Na-acetate 286 Na-borate 177, 178 Na3-citrate x 2H2O 286 Na2HPO4 23, 114, 216, 425 Naked siRNA delivery 215 NaN3 178 Nanodrop 217, 401, 410 Nanodrop Spectrophotometer .217, 401, 410 Nanometer 284 Nanoparticles 190, 216, 219–220 Nasopharyngeal carcinoma (NPC) 44 Natural killer (NK) cells 10, 49 ND filters 274, 278, 280 Nef 54 Nef region 54 Nematodes .5, 6, 8, 12–13 Neocortex 320 Neomycin (Neo) 110, 115, 346 Neuroblastoma 333 Neurodegenerative disease 211–227 Neurogenesis 320 Neurospora crassa Neurotransmitters 214 NF-kB1 344, 358 NF-kB2 344, 358 Ni-NTA agarose 273, 275, 277 NLDC-145 176, 178, 179, 185 Noble agar 248 Nodaviridae 11 Non-adherent cells 180 Non-coding RNAs (ncRNAs) 35–59 Nonconserved miRNAs 40 Non-dividing 320 Non-essential amino acids (NEAA) 402, 416 Nonsense-mediated mRNA decay 38, 114 Normocin 285 Northern blotting .38, 89, 120, 126–130, 290–292 Notch ligands 330, 336 NTS2 387, 388 Nuclear factor kB (NF-kB) 344 Nuclease-free 246–248, 251, 252, 254–256, 267, 284, 285, 290 Nucleases 13, 53, 97, 155–157, 175, 194, 246–248, 251, 252, 254–256, 267, 277, 284, 285, 316, 317, 384, 403 Nucleocapsid (NP) 398, 411 Nucleolytic 317 Nucleolytic degradation 317 Nucleotides (NT) 3, 5, 9, 13, 22, 23, 28, 32, 35, 36, 38, 41, 42, 52, 54, 56, 68, 70, 71, 85, 93, 94, 96–102, 104, 114, 126, 127, 131, 134, 138–140, 145, 147, 150, 151, 167, 212, 214, 225, 243, 247, 293, 315, 326, 327, 384, 411, 412 Nylon membrane Hybond 287 O 2’-O-alkylation 155 2’-O-allyl 157 2’-O-aminoethyl-ribooligonucleotides 156 2’-O-aminopropylribooligonucleotides 156 2’-O-butyl 156 Ocular discharge 414 2’-O-dimethylallyl 156 Off-targeting 94 Off-target silencing 68, 104 2’-OH 156 Oligoadenylate cyclase Oligoadenylate synthetase (OAS1) 109 Oligoadenylate synthetases (OAS) 102 Oligodeoxynucleotides (ODN) 174, 344 Oligo dT 23, 29, 177, 184, 347, 350, 360, 363, 410 Oligofectamine reagent 426, 429 Oligomer cleavage 158 Oligomers 157–159, 163, 165, 167, 233 Oligo primer analysis 95, 101 Oligoribonucleotides 24, 156 Oligos .110, 111, 116, 164, 166, 345, 435 OligoWalk 95, 139, 141–145, 147, 152 2’-O-methoxyethoxy 156 2’-O-methyl 104, 155, 156, 316, 398, 403 2’-O-methyl modifications 104, 316 Omohyoid muscles 418 Oncogene 232, 234, 245 Oncogenic 46, 50, 58, 174, 231–242 Oncoprotein 244, 247, 248, 255–265, 329, 330, 332 One-Step Reverse Transcription-PCR System 425, 428 Open reading frame 73 (ORF73) 50, 54, 96 3’-O-phosphoramidites 160 Ophthalmic lubricant 192 Opioid 383, 384 Optical density (OD) 117, 164–167, 178, 218, 219, 224, 410, 418 Opti-MEM 23, 28, 69, 75, 78, 375, 376 ORF71 See v-Flip Osteopontin (SPP1) 50 Ovalbumin (OVA) 361, 366, 367 Overhangs 7, 28, 32, 82, 88, 89, 93, 96, 97, 116, 138, 140, 155, 277, 315, 316 P PA 398, 411 PAE-Akt1 .247, 255–257 PAE-siRNA 252, 253 PageR Gold precast gels 301 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) 175, 178 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) 175, 178 Paraformaldehyde 217, 222, 360, 386, 392 Parkinson’s disease 320 Passenger strand 7, 37, 126, 137, 140 Pathogenesis 9, 44, 45, 49, 55, 57, 344 PAZ domain 36 PB1 398, 411 PB2 398, 411 PCR reverse primer 184, 364 PD-1 330, 333, 334 PD-L2 330 pDNA 244, 245, 247, 265, 267 pEGFP-N2 247 PEI-complexed siRNA 292, 294 PEI F25-LMW 284, 287, 288, 292, 295 PEI-mediated delivery of nucleic acids 295 PEI/siRNA 284–289, 291–294 PEL-derived cell lines 50 p24 ELISA assay 126, 132, 133 Penicillin 69, 112, 117, 124, 176, 180, 182, 186, 198, 201, 206, 232, 274, 343, 359, 399, 400, 402, 414, 416 Penicillin-streptomycin mix (Nacalai Tesque) .112, 113, 198, 200, 201, 206, 273 Peptides (polyplex) 175 PeqGOLD TriFas 285 Perforin 342 Peripheral blood mononuclear cells (PBMCs) 22, 24–27, 31, 32, 58, 415, 416 Perl script 150 pET-TatU1A-C 273, 275 Petunia pGL3-TK 68, 69, 72, 74, 75, 77 Pharmacodynamic 155, 159 Pharmacokinetic 155, 159, 190, 284, 292 Pharmacokinetic parameters 284 Pharmacopoeia 383 Phenol 112, 198, 202, 285, 290, 412 Phenotype 5, 104, 215, 332, 357, 424 Phenotypic screening 197, 207 Phenylmethylsulfonyl fluoride (PMSF) 113, 217, 224, 233, 273, 300, 426 RNA Interference 445 Index Phosphate buffered saline (PBS) 22, 23, 27, 29, 69, 76, 112–114, 117–119, 178, 180, 182, 185, 186, 198–204, 206–208, 216–218, 233, 239, 247, 248, 255, 258, 259, 263, 285, 293, 294, 300, 302–304, 343, 344, 346, 347, 350, 351, 353, 354, 360, 367, 369, 376, 378, 399, 400, 402, 405–409, 411, 414–416, 418, 420, 425, 427, 428, 432 Phosphitylation 160–162 Phosphoramidite 157–163 Phosphorothioate 155, 156, 167, 316 Phosphorylation 53, 236, 333 Phosphotungstic acid solution 247, 253 Photinus 68–72, 74, 76, 77 Photoinduced RNAi 271–280 Photoinducible 271, 272 Photostimulation 272, 274, 276–280 phRL-TK 68, 69, 72, 74, 75, 77 PI3K 231, 232, 236, 237 p-iodonitrotetrazolium 248, 261 4-Piperazinediethanesulfonic acid (PIPES) 232 Piwi-interacting RNAs (piRNAs) 36 (32P)-labeled material 296 Plaque assay 399–400, 405–406 Plasmacytoid 329, 357 Plasmacytoid dendritic cells 24, 329, 357 Plasmid 4, 43, 68, 69, 72–75, 77, 78, 110–112, 115, 116, 119, 124, 127, 189, 190, 197, 238, 247, 254, 255, 273, 295, 317, 319, 332, 343, 344, 346, 354, 399, 403, 404, 427, 431, 432, 435 Plasmid DNA .69, 73–75, 78, 112, 116, 119, 190, 197, 238, 295, 332, 344, 346, 403, 404, 431, 432, 435 Plasmid pET-TatU1A-C 273, 275 Plasticity related gene (SRGN/PRG1) 50 Pol III promoters 124, 126 Pol II polycistronic transcript 124 Polyamidoamine dendrimers 216, 219 Polyarginine 408 Poly b-amino esters (PAEs) 244–247, 249–257, 265–267 Polycarbonate membrane 176, 179, 233 Polycation 216, 220, 266, 408 Poly (ethylene glycol) diacrylate (PEGDA) 246, 249–250, 265, 266 Polyethylene glycol (PEG) .69, 75, 175, 178, 179, 216, 219, 244, 246, 250, 251, 266, 321 Polyethyleneimine (PEI) 215, 219, 220, 244, 246, 249–251, 265, 266, 283–296, 318, 408, 427, 432 Polyglutamine disorders 321 Poly-L-lysine 219 Poly L-lysine-coated glass slides 199, 202 Polylysine (PLL) 216, 219, 408 RNA Interference 446 Index Polymerase chain reaction (PCR) 22, 23, 28–31, 40, 68, 72, 89, 110, 113, 117, 118, 120, 177, 183–186, 200, 204, 224, 232, 257, 285, 290–292, 299, 308, 343, 347–351, 355, 359–360, 363–365, 387, 388, 390, 394, 400, 401, 405, 409–413, 420, 425, 428–430, 432–434 Polymerases 8, 22, 28, 36, 37, 45, 49, 56, 99, 110, 111, 113, 115–117, 184, 285, 291, 315, 335, 348, 397, 401, 411, 413 Polymerase subunits 398 Polymer-based siRNA delivery 243–268 Polymeric 257 Polyomavirus 51–53 Polyplexes 244, 246–247, 251, 253–258 Poly-Prep chromatography 273 Post-mitotic 320 Post-transcriptional gene silencing 3–7, 10, 93, 137 Post-transcriptional regulators 35, 56 Potato virus X (PVX) 14, 15 Potyvirus Y (PVY ) 15 Precursor miRNA (pre-miRNA) 7, 36, 37, 39, 40, 52, 126, 315 Pre-synaptic protein 59 Primary effusion lymphoma (PEL) 49, 50 Primary miRNAs (pri-miRNAs) 36, 37 Primate foamy type (PFV-1) 8–10 pRNATU6.1, 343–344, 346, 354 Processing bodies (P-bodies) 37, 38 Programmed death-1 ligand (PD-L1) 330, 336 Progressive multifocal leukoencephalopathy (PML) 51, 52 Propidium iodide 248, 259–260, 427 Protease 54, 118, 119, 217, 224, 233, 300, 328 Proteasomes 328 Protein Assay Kit 273, 276 Protein G 176, 178 Protein kinase R (PKR) 8, 53, 55, 56, 58, 102, 109, 140, 316 Protonation 284 Protozoa 314 PstI-KpnI fragment .111, 115, 116 PUMA mRNA 43, 47 Puromycin 427, 431 PVDF membrane 234, 240, 301, 305, 306 Pyronin-Y 343 Q qRT-PCR ELISA 285, 290–292, 410 Quantitative real-time polymerase chain reaction (qRT-PCR) 285, 290 Quelling 3, R RACE assay kit 401 Radioactive labelling 134 Radio immuno precipitation assay (RIPA-Lysis) buffer 217–218 Raf-1 317 Random hexamer primer 285, 291 Rapid amplification of cDNA ends (RACE) 52, 401–402, 409, 411 RAW cells .25–26, 31, 32 RAW 264.7 cells 24, 25 rde-1 13 rde-4 13 Real-time machine 177 Real-time PCR 29, 120, 173, 177, 183–186, 299, 308, 343, 347–351, 355, 359, 363–365, 390, 394, 400–401, 409, 411, 425, 432–434 Real-Time SYBR green PCR system 425 Recombinant GM-CSF 176, 180, 359 Reference dye 177, 285, 360, 364 Regulatory T cells (Treg) 331, 357, 358, 361, 368–369 RelA 344, 358 RelB 344, 346–347, 358–362, 365–369 Renilla luciferase 67–70, 76, 77, 404–405 Repeat-associated small interfering RNAs (rasiRNAs) 36 Reporter alleles 67–70, 72, 74, 76–78 Reporter protein (EGFP) 244, 247, 253–254 Restrains 191, 220 Retinoblastoma 428 Retinoic acid inducible gene I (RIG-I) 21 Retinoic-acid-inducible protein Retrotransposon Retroviral coat proteins (env) 54 Retrovirus 8, 9, 12, 42, 53, 55 Reversed-phase (RP) 164, 165 Reversed-phase HPLC 164 Reverse transcriptase (RT) 23, 39, 173, 177, 183–185, 285, 291, 347–350, 360, 425 Reverse transcription-polymerase chain reaction (RT-PCR) 22, 28, 30, 40, 89, 110, 113, 117–118, 120, 184, 204, 290, 343, 348, 349, 351, 405, 425, 428–430 Reverse transfection 28, 197–209 Reversible permeabilization 302–305, 307 Revert Aid H Minus M-MuLV reverse transcriptase 285 Reynolds rational rules 97 RFF-3 13 Rhodamine 247, 255–257, 268 Ribonuclease (RNase) III 36 Ribonucleo protein 7, 36, 37, 157, 163, 274, 315, 358 RNA Interference 447 Index Ribonucleoprotein complexes 315 Ribonucleoside phosphoramidites 157 Ribophosphoramidites 157 Ribosomal RNA (rRNA) 117, 410 Riboviruses 11 Ribozymes 138, 342 RIG-1 8, 140 RIPA buffer 113, 118, 224 RISC-mediated cleavage 145 RNA-binding protein (RBP) 36, 272 RNA-binding protein kinase R (PKR) 53, 102, 109, 140, 255, 316 RNA decapsidation 54 RNA degradation 410 RNA-dependent RNA polymerase (RdRp) 8, 11–13, 15 RNA extraction 23, 29, 113, 133, 183, 217, 222, 226, 435 RNAi Codex 103 RNA-induced silencing complex (RISC) .6, 7, 11, 13, 56, 93, 96, 99, 101, 102, 126, 137–140, 144, 145, 155, 174, 243, 245, 315, 326, 327, 358, 384, 403, 409, 411, 412 RNA-induced silencing complex (RISC/RITS) 7, 56, 137, 315, 358 RNA interference (RNAi) 3, 67, 81, 109, 137, 214, 283, 314, 326, 358, 383, 398, 418 RNAi pathway 6, 11, 15, 101, 147, 214 RNA isolation 177, 290, 347, 359, 363, 401, 409, 411 RNA-mediated gene silencing 13, 38 RNA oligomers .163, 165, 233 RNA-oligonucleotides 155 RNAplfold .144, 145, 150 RNA polymerase II 36, 37, 57, 99, 110, 335 RNA polymerase III 56, 99, 110 RNase 23, 29, 36, 39, 52, 68, 102, 169, 187, 190, 217, 223, 224, 247, 265, 274, 276, 277, 285, 290, 291, 300, 303, 308, 315, 316, 347, 355, 363, 375, 379, 400, 401, 410, 412, 420, 427 RNAse A/T1 digestion 39 RNase AWAY® Reagent 400, 420 RNase-free buffer TE 217 RNase-free water 169, 187, 223, 224, 274, 285, 290, 300, 400, 401, 410 RNase III 3, 7, 36, 93, 175, 179, 315, 326, 358 RNase inhibitor 177, 285, 291, 347, 360, 363 RNAseL/PKR RNase protection assay (RPA) 39, 52 RNaseZap® 217, 222, 300, 303, 308 RNA spreading defective (rsd) 12 RNAxs 139, 144–150, 152 RNeasy minelute kit 217, 223 Roseolovirus 51 Rostro ventral medulla 387 RP-HPLC 164–165 RPMI 1640 22, 124, 176, 180–182, 185, 186, 232, 233, 238, 248, 254, 255, 258, 260, 264, 300, 302, 303, 343, 346, 347, 359, 361, 362, 365–368 rrf-1 12 RSS Tas S S100 calcium binding protein A2 (S100A2) 50 SDS-PAGE protocol .224, 239–240 SDS-polyacrylamide accumulative gel 426 Segregation Sephanous vein 221 Sepharose 176, 178, 180 shGFPU1A 279 ShortCut RNase 175, 179 Short hairpin RNA (shRNA) .8, 53, 68, 95, 103, 104, 109–121, 123, 124, 126–127, 243–268, 271, 272, 274, 276–279, 295, 316, 320, 321, 329, 345, 346, 350, 398, 403, 431, 432 Short interfering RNA (siRNA) 3, 21, 36, 67, 81, 93, 109, 124, 137, 155, 173, 189, 197, 211, 231, 243, 258, 271, 283, 299, 314, 326, 341, 373, 383, 398, 424 ShRNA-encoding 295 sid-1/rsd-8 12 Signalling pathways 47 Silence® Negative Control siRNA 199, 237, 369 Silica gel 157, 161, 162, 208 Simian immunodeficiency virus (SIV) 54 Simian virus 12 (SV12) 41, 52 Simian virus 40 (SV40) 41, 51 Sindbis virus (SINV) 11 Single nucleotide polymorphism (SNP) 85, 96, 97 siRNA amplification 8, 11–13, 291, 319, 411 siRNA biodistribution 190, 286–287, 292–294 siRNA compaction (‘condensation’) 284 siRNA delivery .24, 168, 175, 190, 194, 211–227, 243–268, 271–280, 284, 287, 292, 293, 317, 376 siRNA duplex 3, 7, 28, 70–72, 75, 96, 99, 102, 104, 138, 139, 168, 190, 191, 194, 300–305, 307, 308, 315, 374–377, 424, 429–431 siRNA-nanocomplex 220–221 siRNA_profile 96, 97, 99, 100, 103, 104 siRNA protocols 243 siRNA transfection 91, 181–182, 205, 215, 248, 288–290, 304, 305, 359, 361, 367, 370 Smad 333 RNA Interference 448 Index Smad 10, 333 Small temporal RNA (stRNA) SNAP25 51, 59 snoRNA 126, 127 SNP-based filtering 88 snRNAs 156 SOC medium 273, 275 Sodium chloride (NaCl) .23, 68, 69, 113, 114, 157, 158, 161, 162, 216, 233, 234, 247, 256, 273, 274, 284–286, 292, 300, 301, 384, 385, 425–427 Sodium dodecyl sulfate (SDS) 113, 114, 118–119, 125, 233, 300, 301, 343, 411, 426 Sodium hydrogencarbonate 161 Sodium orthovanadate 217 Sodium Pentobarbital (Nembutal) 192 Somatic cells Sonication 273, 277 Spectrophotometer .176, 217–219, 224, 256, 401, 410 Spectroscopy 166, 246, 250 Spermidine 296 Sph I 68, 70, 71, 73, 74, 78 Spinocerebellar ataxia type 321 Splicing 7, 81–91, 95–97, 425 SSC buffer 286, 294 Stacking buffer 234, 240 Standard Northern blot hybridization 38 STAT1 329 Stereotaxic intra-cerebroventricular injection 329 Sterilized MilliQ water 376 Streptavidin-horseradish peroxidase (SAv-HRP) 27, 132, 135 Streptolysin-O (SLO) 299, 300, 302–305, 307, 308 Streptomycin 69, 112, 124, 176, 180, 182, 186, 198, 201, 206, 232, 274, 343, 359, 399, 400, 402, 414 Stripping buffer 301, 306 Stylohyoid muscles 418 Sugar moiety 160 SuperScript III First-Strand Synthesis System 23, 29, 401, 410 Superscript II Reverse Transcriptase 113, 117, 177, 184, 360, 363 Supine 193 Suppressor of cytokine signaling (SOCS1) 329, 336 SYBR Green 23, 29, 177, 184, 285, 291, 343, 348, 350, 360, 364, 401, 411, 425 SYBR PrimeScript RT-PCR Kit 200, 204 Synergism 15 Synthetic oligonucleotides 70, 71, 155 Systemic administration 189, 316, 317, 321 Systemic injection 350 Systemic RNAi defective (sid) 12 T 293T 43, 124, 236, 242, 375–377 T antigens 52 Taq DNA polymerase 113, 117, 184, 401, 413 TaqMan Universal PCR Master Mix 401 TAR element 39, 55 Tat-peptide 272 TatU1A 271–280 TatU1A-Alexa 271, 272, 275–276, 278–280 TBST buffer 426, 429 Tc1 12 T cell proliferation 186, 334, 336 T cell receptor (TCR) 328, 373 T cells 48, 50, 54, 58, 124, 133, 173, 174, 186, 326–336, 358, 373, 374 T4 DNA ligase 343 Terminal deoxynucleotidyl transferase (TdT) .343, 352, 413 Terminus 97, 99, 100, 163, 193 Tetrahydrofurane 161 Tetrahymena Tetramethyl benzidine (TMB) 23, 27, 417 T98G cells 22, 28–30 TGF-b receptor (TGF-b R) 333 Th1 326, 329, 331–332, 358 Th2 331, 332, 374 Th17 331 Thermodynamic asymmetry 139 Thermodynamic factors 99 Thiolation 178, 187 Th1-mediated immunity 332 Th1 T cell 358 Thymidine kinase 49, 69, 70 Thymidylate synthetase 49 Tiny non-coding RNAs (tncRNAs) 36 Tissue Culture Infective Dose 50 (TCID50) 405–407, 409 Tissue homogenization 222–224 Tlc silica gel F254 157 TLR3 21 TLR7/8 .21, 22, 24, 103 T lymphocytes 333 Tobacco etch virus (TEV) 14 Tolerogenic DCs (tol-DCs) 174, 358 Toll-like receptors (TLRs) .7, 21, 25–27, 140, 331, 379 Topical application 373–380 T7 polymerase 22 T4 polynucleotide kinase 286, 293 TRAF6 331 TRAIL 200, 206 RNA Interference 449 Index Tranfection reagent 177 Transactivating responsive (TAR) 36, 39, 55, 126, 127 Transcriptional inhibition 4, 16 Transcriptome .120, 214, 434 Transduction buffer (Buffer-T) 273, 275, 276, 278, 279 Transfection complex 202, 203 Transfection microarrays (TMAs) 198–199, 201, 202, 206, 207 Transforming growth factor-b (TGF-b) 10, 333 Transgenes 4, 5, 11, 12 Translation 35, 59, 94, 97, 139, 314, 334–336, 383 Translational repression 7, 16, 37, 214, 327 Transposons 4, 7, 8, 12, 314 Treg cells (CD4+CD25+ FoxP3+) 358 Triethylamine (NEt3) 157 Triethylammonium acetate 164 Trigeminal ganglia 43 5’-Triphosphates 22, 27, 28 Triple-shRNA expression vector 110, 115, 116, 118, 120 Tris-acetate-EDTA (TAE) buffer 69, 74, 246, 251–253 Tris-HCl 68, 69, 113, 176–179, 233, 234, 285, 286, 300, 301, 343, 401, 413, 426 Tris-phosphate 286 Triton X-100 285, 300 TRIzol 177, 183, 217, 222, 223, 226, 347, 350, 359, 363, 401, 411, 412, 425, 428 tRNAs 156 Trypan blue 248, 258–300, 302 TryplE-Express 31 Trypsin-EDTA .69, 124, 201, 207, 232, 248, 258, 263, 399 Tryptone 273 Tubulointerstitial nephritis 51 Tumor-associated antigen (TAA) 328 Tumorigenesis 232, 244 Tumor microenvironment .326, 328, 330, 333 Tumor necrosis factor-a (TNF-a) 23, 317 Tumor necrosis factor receptor (TNFR) 46 Tumor-specific immune responses 328 TurboCapture mRNA kit 200, 204 Turnip crinkle virus (TCV) 14 Turnip mosaic virus (TMV) 14 Tuschl’s and Reynold’s rule 212 Tyro3/Axl/Mer (TAM) 329, 336 U U1 58, 274 U6 110, 131, 431 U1A .272, 274, 276, 277 UL15 42 UL15.5 42 UL16 49 UL18 49 UL40 49 UL83 49 UL141 49 UL142 49 a, ß-Unsaturated 249 3’ Untranslated region (3’UTR) 9, 35, 45, 46, 48, 50, 58, 78, 96, 97, 327 Untranslated regions (UTRs) .9, 10, 35, 45, 46, 48, 50, 51, 58, 70, 78, 96, 97, 327 Uranil acetate solution 247, 253 Uridine 22, 24, 160, 162 UV 114, 121, 129, 158, 166, 169, 218, 219, 246, 252, 253, 256, 267, 344 UV-spectrophotometry 166 UV-spectroscopy 166 V Vacuum chamber 202, 203 VAI 41, 46, 52, 53, 55, 56 VAII 41, 46, 52, 53, 56 VAII RNA 46, 53, 56 VAI RNA 52, 53, 55, 56 Vascular endothelial growth factor (VEGF) 320 Vascular endothelial growth factor A (VEGF-A) 232 Vascular thrombosis 350 v-cyclin (ORF72) 50 Vero cells 405 Vesicular stomatitis virus (VSV) 9, 13 v-Flip (ORF71) 50 Viral carriers 317 Viral encephalitis 52 Viral-encoded miRNA Viral RNA transcripts 42, 43 Viral tropism Viral vectors 123, 215, 320, 335 Virus-induced gene silencing 5, 11, 14 W Wallac Betaplate liquid scintillation counter 178, 186 Western blot analysis 113–114, 118–120, 225, 233–236, 238, 240–241, 257, 292, 299, 301–308, 390, 394, 426, 428 West Nile virus 10 Wild-type alleles 71, 76, 77 WST-1 assay 296 RNA Interference 450 Index X Xenograft 286, 292, 424, 428, 432, 435 Xeroderma pigmentosum group A (XPA) 109, 111, 113–115, 118, 120, 121 Xiphoid 193 xMAP 419 XP2OSSV 116 Xylazine 193, 216, 349, 386, 390, 391, 400, 402, 408, 414, 418 Xylenecyanol 286 Z Zwitterionic oligonucleotides 156 ... complementary to the stRNA (20) Particularly, stRNAs lin-4 and let-7 were determined to bind with the 3¢ noncoding regions of target lin-14 and lin-41 mRNAs, respectively, leading to reduction in mRNA-encoded... homology-dependent, posttranscriptional Wei-Ping Min and Thomas Ichim (eds.), RNA Interference, Methods in Molecular Biology, vol 623, DOI 10.1007/97 8-1 -6 076 1-5 8 8-0 _1, © Springer Science + Business... MediStem Laboratories Inc San Diego, CA USA thomas.ichim@gmail.com ISSN 106 4-3 745 e-ISSN 194 0-6 029 ISBN 97 8-1 -6 076 1-5 8 7-3 e-ISBN 97 8-1 -6 076 1-5 8 8-0 DOI 10.1007/97 8-1 -6 076 1-5 8 8-0 Springer New York