Recent Work & Current Methods in Neuroscience microRNA Research

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• Brief review of microRNA basics: history, biogenesis, function • Recent developments of microRNA research in the field of neuroscience • Current methods for microRNA discovery and profiling • Case studies and application examples

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Neuroscience microRNA Webinar Recent Work & Current Methods In Neuroscience microRNA Research Christoph Eicken , PhD Head of Technical Services – Microarrays 2012-10-09

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Agenda Recent Studies Current methods Q & A Case Studies miRNA Intro

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Transcriptomics microRNA Profiling Microarray Services microRNA Discovery Sequencing Services Seq- Array SM Method Proteomics Phosphopeptide Binding Microarray Service Protein- Protein Interaction Microarray Service Kinase Profiling Microarray Service 5 µ Paraflo ® Microfluidic Technology High Density – On Chip Parallel Synthesis Customizable – DNA, RNA, Peptides, and Analogs Quality Data – Microfluidics / Synthesis Chemistry

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A ll miRNAs are small non-coding RNAs, usually consisting of ∼20–22 nucleotides for animals and ∼20–24 nt for plants. All miRNA precursors have a well-predicted stem loop hairpin structure, and this fold-back hairpin structure has a low free energy Many miRNAs are evolutionarily conserved, some from worm to human in animals, or from ferns to core eudicots or monocots in plants Bind to complementary mRNA molecules and act as negative regulators of translation High copy number Expression is tissue (and developmental stage) specific microRNAs - What W e Know

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microRNAs - What W e Know Currently - 25,141 mature miRNAs across 193 plant, animal, and virus species ( miRBase 19 , Aug. 2012). Mechanism is far reaching and complex – each miRNA may control many genes and it is estimated that miRNAs regulate expression of up to 1/3 of all human genes. Operate by one of two hypothesized mechanisms : – Complete pairing mRNA is degraded - predominant in plants – Imperfect pairing translation is repressed but mRNA remains intact - predominant in animals

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1993 – Lin-4 was shown to encode two small RNA molecules (not protein) Small RNAs control developmental timing in C. elegans through negative regulation of lin-14 gene. Lee RC et al. 1993 The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843-54. [ article ] 1998 – RNAi is observed for the first time – leads to Nobel Prize (2006) Fire A, Mello CC et al. 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391:806-811. [ article ] Challenge to the Central Dogma of Biology DNA > transcription > RNA > translation > protein 2000 – Let-7 was identified in humans and Drosophila . (Reinhart et al., Slack et al.) 2001 – Bartel , Tuschl , Ambros - Discover large class of small regulatory RNAs, name them microRNA (miRNA) Lau NC et al. 2001 An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans . Science 294(5543):858-62 . [ abstract ] Lagos-Quintana M et al. 2001 Identification of novel genes coding for small expressed RNAs . Science 294(5543):853-8 . [ abstract ] Lee RC et al. 2001 An extensive class of small RNAs in Caenorhabditis elegans . Science 294(5543):862-4. [ abstract ] 2002 – Identification of Drosha reveals complete microRNA biogenesis pathway pri-miRNAs > Drosha > pre- miRNAs (~70nt) > Dicer > mature miRNAs (~22nt) Lee Y et al. (2002) The nuclear RNase III Drosha initiates microRNA processing. Nature 425(6956):415-9. [ abstract ] Milestones

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2003 – Hobert Lab – discovery that miRNAs govern neuronal cell fates Hobert and colleagues showed that a miRNA-driven feedback loop governs the decision between two neuronal cell fates in C. elegans. Johnston, Hobert 2003 [ abstract ] Show that a previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. ASER and ASEL neurons undergo a shared precursor state (ASE), but differentiate as two functionally distinct neurons. 2003 – Stoffel Lab - First report of in vivo silencing of miRNAs with antagomirs used chemically engineered oligonucleotides Krutzfeldt J et al. (2005) Silencing of microRNAs in vivo with ' antagomirs '. Nature 438(7068):685-9. [ abstract ] 2004 – Microarrays are used to profile miRNA expression Several groups develop or modify existing gene expression microarrays for profiling miRNAs Babak et al. 2004 [ article ], Barad et al. 2004 [ article ], Liu et al. 2004 [ article ], Nelson et al. [ abstract ], 2004, Thomson et al. 2004 [ article ]. Milestones

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2005 – Next-Gen Sequencing is used for small RNA discovery and analysis Green Lab - uses Solexa (now Illumina) sequencing to identify novel small RNAs in Arabidopsis plants Lu C et al. 2005 Elucidation of the small RNA component of the transcriptome. Science, 309: 1567–1569. [ abstract ] 2006 – miRBase goes online at Sanger Institute Depository for experimentally verified microRNAs: http://www.mirbase.org/ Griffiths-Jones S et al. 2006 miRBase: microRNA sequences, targets and gene nomenclature. NAR 34(Database Issue):D140-D144. [ article ] 2008 – Circulating miRNAs detected in body fluids miRNA signatures in CSF, serum and plasma provide cancer diagnosis / prognosis Cerebrospinal - Cogswell et al. (2008) Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways. J Alzheimers Dis 14(1):27-41. [abst r act] Serum - Chen X et al.(2008) Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006. [ article ] Plasma - Mitchell PS et al.(2008) Circulating microRNAs as stable blood-based markers for cancer detection. PNAS USA 105:10513–10518. [ article ] Milestones

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2005 - MicroRNA in Glioblastoma Croce Lab – had first report about the role of miRNAs in cancer in 2002. PNAS 99, 15524–15529. [ article ] Chan et al. 2005 MicroRNA-21 Is an Anti-apoptotic Factor in Human Glioblastoma Cells. Cancer Res 65(14):6029-33. [ article ] Ciafre ` et al. 2005 Extensive modulation of a set of microRNAs in primary glioblastoma . BioBio Res Com 334:1351–1358. [ article ] 2005 - Tourette’s syndrome The first psychiatric disease linked to miRNA expression and function. Abelson et al. 2005 Science 310(5746), 317-20. [ abstract ] Mutations in the 3′UTR of SLITRK1 were affecting the targeting by miR-189. 2006 - Brain development The first demonstration that miRNAs are localized in the synapse and can regulate dendritic spine structure -miR-134 Schratt et al. 2006 A brain-specific microRNA regulates dendritic spine development. Nature 439(7074), 283-9. [ abstract ] 2007 - Schizophrenia, Alzheimer’s disease Rogaev et al. 2007 MicroRNA in Schizophrenia, Biochemistry ( Mosc ) 72(5), 578-82. [ article ] Perkins et al. 2007 microRNA expression in the prefrontal cortex of individuals with schizophrenia and schizoaffective disorder. Genome Biol 8(2), R27. [ article ] Lukiw WJ. 2007 MicroRNA speciation in fetal, adult and Alzheimer's disease hippocampus. Neuroreport 18(3), 297-300. [ abstract ] Milestones - Neuroscience

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2008 – Autism Talebizedah et al. 2008 Autism Research 1(4), 240-50. [ article ] 2008 – Prion-based Disorders Saba et al. 2008 A miRNA signature of prion induced neurodegeneration. PLoS One 3(11),e3652. [ article ] Lukiw et al. 2011 Up-regulation of -miRNA-146a, a marker for inflammatory neurodegeneration, in sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker (GSS) syndrome. J Toxicol Environ Health A 74(22-24), 1460-8. [ abstract ] 2009 – Traumatic Brain Injury Redell and Dash. 2009 Traumatic brain injury alters expression of hippocampal microRNAs: potential regulators of multiple pathophysiological processes. J Neurosci Res 87(6), 1435-48. [ abstract ] 2010 – Astrogliosis Pogue et al. 2010 Micro RNA-125b (miRNA-125b) function in astrogliosis and glial cell proliferation. Neurosci Lett 476(1), 18-22. [ abstract ] Milestones - Neuroscience

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pri-miRNA = primary microRNA transcript pre- miRNA = precursor microRNA miRNA * = antisense microRNA (now -3p or -5p) miRISC = microRNA-induced silencing complex For latest information regarding miRNA nomenclature see the miRBase.org blog . miRNA Processing

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miRNA Biogenesis Pathway (A) Animal and (B) plant miRNA biogenesis. Mature miRNAs are indicated in red and miRNA* strands are in blue. Du T, Zamore PD. 2005 microPrimer : the Biogenesis and Function of microRNA. Development 132: 4645-4652. [ article ]

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miRNAs in miRBase 21,264 miRBase Entries - 193 plant, animal, and virus species miRBase: microRNA sequences, targets and gene nomenclature. Griffiths-Jones S et al. NAR 2006 34(Database Issue): D140-D144 [ article ]

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miRNAs in PubMed

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Why Study miRNAs in Neuroscience? A large number of studies have identified miRNAs that are important for brain development and neuronal differentiation. (Li and Jin, 2010 ; Bian and Sun, 2011 ). miRNAs have been proven to influence brain maturation and plasticity, mechanisms that are known to be perturbed in psychiatric diseases. ( Mellios and Sur, 2012 [ article ]) microRNAs play roles in multiple hallmark biological characteristics of glioblastoma, including cell proliferation, invasion, glioma stem cell behavior, and angiogenesis. ( Lawler and Chiocca, 2009 [ abstract ]) .

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miRNAs in Neuroscience: Clinical Potential Molecular Diagnostics / Biomarkers – Identification of specific miRNAs or miRNA expression based signatures that can act as biomarkers for various diseases/pathologies. Biomarkers in body fluids: plasma, exosomes, cerebrospinal fluid Make accurate and detailed clinical diagnosis Potential to determine prognosis and predict treatment efficacy Rosetta Genomics' miRview Lung Assay: Assay Shown to Accurately Differentiate Between the Four Main Types of Lung Cancer

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Drug Discovery / Therapeutics – Identification of miRNAs that play essential roles in disease to act as drugs or possible therapeutic targets. miRNAs as drug targets miRNAs as therapeutic agents In addition to mimetic and antisense oligonucleotides to modulate miRNA function, recent efforts have also been directed toward developing small-molecule drugs that can influence the biogenesis of miRNAs or directly influence their function miRNAs in Neuroscience : Clinical Potential

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In fully differentiated neurons, miRNAs are localized at dendritic spines and postsynaptic densities. Expression of many miRNAs is dynamically regulated during neurogenesis, neuronal maturation and brain development. miRNAs can maintain plasticity-related genes in translational repression until alleviated by neuronal activity MicroRNA expression is altered in many neurodevelopmental disorders such as fragile X syndrome, rett syndrome & autism. MicroRNA expression is altered in many neuropsychiatric disorders such as depression, schizophrenia & drug addiction. Im HI, Kenny PJ. (2012) MicroRNAs in neuronal function and dysfunction . Trends Neurosci 35(5), 325-34. [ abstract ] 2012 Review: MicroRNAs in neuronal function and dysfunction MicroRNAs play an essential part in all aspects of neuronal development, function and plasticity. Dysfunction in miRNA signaling contributes to neurodevelopmental & neuropsychiatric disorders

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Disorder miRNAs Proposed contribution to disorder Refs Fragile X syndrome miR-124 FMRP may regulate miR-124 levels in adult brain. [57] miR-9, miR-124, miR-125a,b, miR-128, miR-132, miR-219 (i) FMRP is bound to these miRNAs. (ii) Thinner dendritic spines are promoted by miR-125, and the opposite effect is induced by miR-132. The effects of both miRNAs are dependent upon FMRP. [38] Rett syndrome miR-132 Negative feedback loop between MeCP2 and miR-132. Disruption of homeostatic interactions between these factors may contribute to RTT symptomatology. [62] and [63] let-7a, miR-7a*, miR-7i, miR-9*, miR-15a*, miR-24, miR-28, miR-29a*, miR-30d, miR-34b-3p, miR-34c, miR-101a, miR-106a, miR-124, miR-137, miR-206, miR-296-5p, miR-299*, miR-323-3p, miR-326, miR-328, miR-377, miR-455, miR-495, miR-543, miR-666-5p, miR-674, miR-744*, miR-874 miR-137, miR-187, miR-197 miR-132, miR-212 miR-29b, miR-92, miR-329, miR-199b, miR-296, miR-221, miR-382 miR-184 MeCP2 represses the expression of these miRNAs [104] [105] [61] , [62] , [63] and [104] [106] [107] miR-197, miR-199a, miR-221 miR-122a, miR-130, miR-146a,b, miR-342, miR-409 miR-7a*, miR-29c, miR-140 MeCP2 increases the expression of these miRNAs. [105] [106] [104] miR-132, miR-212 miR-155, miR-802 miR-302 miR-130b MeCP2 expression is regulated by these miRNAs. [61] , [62] , [63] , [104] and [108] [109] [110] [111] Autism spectrum disorder (ASD) miR-132 Downregulated in ASD postmortem brain. [64] miR-23a,b, miR-25, miR-29b, miR-30c, miR-93, miR-103, miR-106b, miR-107, miR-185, miR-186, miR-191, miR-194, miR-195, miR-205, miR-342, miR-346, miR-376a-AS, miR-451, miR-519c, miR-524 Upregulated in lymphoblastoid cell lines derived from ASD patients. [112] miR-16-2, miR-106b, miR-132, miR-133b, miR-136, miR-139, miR-148b, miR-153-1, miR-189, miR-190, miR-199b, miR-211, miR-219, miR-326, miR-367, miR-182-AS, miR-455, miR-495, miR518a, miR-520b Downregulated in lymphoblastoid cell lines derived from ASD patients. [112] miRNAs in Neuroscience: MicroRNA alterations in neurodevelopmental disorders

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miRNAs in Neuroscience: MicroRNA alterations in neuropsychiatric disorders Disorder miRNAs Proposed contribution to disorder Refs Depression miR-182 miR-30e May increase the risk of major depressive disorder. [66] [67] miR-16 Negatively regulates the expression of SERT, and hence may alter responsiveness to antidepressant treatments. [68] miR-96 Negatively regulates the expression of 5-HT1B receptor. [101] miR-18a Increased expression in the hypothalamic paraventricular nucleus of F344 rats with increased vulnerability to stress. [113] Schizophrenia (Ref. [114] for a more comprehensive list). miR-7, miR-106 let-7d, miR-7, miR-15a,b, miR-16, miR-20a, miR-26b, miR-27a, miR-29c, miR-31, miR-33, miR-101, miR-105, miR-107, miR-126*, miR-128a, miR-153, miR-181a,b,d, miR-184, miR-195, miR-199a, miR-219, miR-223, miR-302a*, b*, miR-338, miR-409-3p, miR-512-3p, miR-519b. miR-17-5p, miR-25, miR-92b, miR-105, miR-134, miR-148b, miR-150, miR-152, miR-154, miR-187, miR-193a, miR-199a*, b, miR-222, miR-328, miR-382, miR-409-3p, miR-423, miR-425-5p, miR-433, miR-452*, miR-487a, miR-495, miR-502, miR-512-3p, miR-519c, miR-532, miR-542-3p, miR-548b, miR-590, miR-592, miR-652, miR-767-5p. miR-7, miR-34a, miR-132, miR-132*, miR-154*, miR-212, miR-544. miR-22, miR-27b, miR-106b, miR-138, miR-148b, miR-151, miR-193b, miR-210, miR-301, miR-324-3p, miR-338, miR-339, miR-425, miR-545, miR-639. These miRNAs are dysregulated in postmortem PFC from schizophrenia patients. [71] and [115] [70] [102] and [116] miR-181b Upregulated in the superior temporal gyrus and dorsolateral PFC of schizophrenia patients. [69] , [70] and [71] miR-219 Expression is disrupted in the PFC of human schizophrenia patients. Exerts a tonic inhibitory influence on cortical NMDA receptor signaling. Expression in PFC of mice is decreased by psychotomimetics. Decreasing miR-219 signaling in brain attenuated schizophrenia-like behavioral deficits in mice. [71] [80] Drug addiction let-7c,f, miR-22, miR-23b, miR-30b,d, miR-124a, miR-125b, miR-126, miR-128b, miR-129*, miR-132, miR-191, miR-212, miR-219, miR-328, miR-329, miR-338, miR-352, miR-376b, miR-383 Altered expression in dorsal striatum after cocaine self-administration in rats. [43] and [62] miR-7a, miR-130a, miR-136, miR-137, miR-138, miR-148b, miR-154, miR-181a, miR-186, miR-301b, miR-324-5p, miR-337-3p, miR-369-3p, miR-376a,c, miR-380-3p, miR-384-5p miR-467a,b, miR-488, miR-500, miR-544, miR-665 Increased expression in D2 receptor-expressing cells of striatum after cocaine treatment of mice. [6] miR-29a,b, miR-34c, miR141, miR-154, miR-182, miR-183, miR-190b, miR-200a,b,c, miR-217, miR-298, miR-329, miR-380-3p, miR-381, miR-429, miR-467c,e, miR-496, miR-500, miR-504, miR-582, miR-680, miR-685, miR-743b, miR-874 Altered expression in midbrain and PFC in mice receiving injections of nicotine, cocaine or amphetamine. [103] let-7d, miR-124, miR-181a. Cocaine alters the expression of these miRNAs in striatum and forebrain of mice, and in turn these miRNAs influence cocaine reward. [117] and [118] miR-9 May contribute to tolerance to the physiological and behavioral actions of alcohol. [85] miR-23b Negatively regulates the expression of the μ opioid receptor. Expression is increased in cultured cells after chronic morphine exposure. [119] and [120] let-7 Negatively regulates μ opioid receptor expression and may therefore regulate the development of opioid tolerance. [121] miR-15b, miR-16, miR-21, miR-23a,b, miR-24, miR-26a,b, miR-30c, miR-99b, miR-103, miR-107, miR-132, miR-146a, miR-150, miR-155, miR-181b, miR-191, miR-221, miR-320a,c, miR-423-5p, miR-638, miR-1469, miR-1826, miR-1915 Morphine alters the expression of these miRNAs in human macrophages. [122] miR-190 Decreased expression in cultures of hippocampal neurons of rats and mice after long-term treatment with the μ opioid receptor agonist fentanyl but not after morphine treatment. [123] , [124] and [125] miR-16, miR-21, miR-140* Nicotine treatment increases the expression of these miRNAs in cultured cells. [126] and [127] miR-133, miR-590 Nicotine decreases the expression of these miRNAs in atrial tissues. [128]

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miRNAs in Neuroscience: microRNA ID Expression Phenotype Targets References miR-21 High Reduced growth (in vitro and in vivo) Apoptosis induction Reduced invasion PTEN, TIMP3 RECK , PDCD4 TMP1 [ 47 , 50 – 56 ] miR-7 Low Reduced growth (in vitro) Reduced invasion EGFR , IRS2 PAK1, SPATA2 [ 57 ] miR-124 Low Reduced growth (in vitro) Neuronal differentiation SCP1, CDK6 PTBP1, ITGB1 LAMC1 [ 48 , 58 – 60 ] miR-137 Low Reduced growth (in vitro) Neuronal differentiation CDK6 , MITF [ 48 ] miR-128 Low Reduced growth (in vitro and in vivo) Reduced stem cell renewal EGFR, BMI1 E2F3A [ 49 , 64 , 65 ] Validated glioblastoma microRNAs with potential functional importance Lawler S, Chiocca EA. (2009) Emerging functions of microRNAs in glioblastoma. J Neurooncol 92(3):297-306.

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miRNAs in Neuroscience: Mellios N, Sur M. (2012) The Emerging Role of microRNAs in Schizophrenia and Autism Spectrum Disorders. Front Psychiatry 3, 39. [ article ] MicroRNAs involved in brain plasticity and maturation with links to schizophrenia and autism spectrum disorders.

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MicroRNA profiling: approaches and considerations Colin C. Pritchard, Heather H. Cheng & Muneesh Tewari Nature Reviews Genetics 13, 358-369 (May 2012 ) .[ abstract ] MicroRNA Profiling Review:

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Seq-Array SM ACGT101-miR Stem-Loop Specific Detection & Profiling Northern Blotting In situ hybridization Real-time PCR Microarray analysis Next-gen Sequencing Target Determination Bioinformatics – TargetScan , PicTar , PITA Gene / proteome expression analysis Pull-down assays 5′ RACE analyses Degradome Sequencing Functional Analysis Lucifierase Assays Gene knockout/overexpression models miRNA inhibition - antagomirs miRNA mimicry Degradsome Seq Digital Gene Expression Pathway Network miRNA Identification Genetic screening Direct cloning, sequencing Computational strategy – MIRCheck , findMiRNA , MIRscan , MiRAlign Tiling Microarrays Next-gen Sequencing Pathway Analysis Bioinformatics – miRFocus , mirPath , MMIA microRNA Research Tools

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miRNA microarray qRT -PCR custom miRNA microarray Next Gen Sequencing Discovery Profiling Quantitation Validation 3 Major Steps & Technologies } Seq-Array SM

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Sample Preparation Cell Line Tissue Blood Serum Plasma FFPE Block Algea Plant Material Fatty Tissue (Viscous Samples) Total RNA (1-4 µg) Norgen Biotek (phenol free) Ambion Qiagen S elect a kit designed to retain small RNA Select kit based on your sample type Use the same kit for entire experiment miRVana Kit miRNeasy Kit Total RNA Extraction Kit Urine

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You can check the UV spectrum of your sample with a spectrophotometer. ↑ 1.0 ↑ 1.8 260nm 230nm 260nm 280nm Bioanalyzer or 1-1.5% agarose gel 28S rRNA band at 4.5kb - ~2X intensity 18S rRNA band at 1.9kb. For Average Cell Line or Tissue sample – RIN number must be ↑ 7 For other sample types such as Blood or Plant – RIN number does not apply Excessive smearing on the gel indicates degraded RNA. Customer Sample Quality Control

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Customer Sample Quality Control microRNA Microarray Expression Profiling Good Poor Good Poor Good Poor Failure in recovery of RNA <200 nt (including microRNA) 1.5% Formaldehyde Agarose Gel Agilent BioAnalyzer Gel Image Urea-PAGE Gel Images © Norgen Biotek

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Biological Replicates – Still Very Important For experiments performed with a small number of biological replicates, significant results may be due to biological diversity between individuals and may not be reproducible - it is impossible to know whether expression patterns are specific to the individuals in the study or are a characteristic of the test condition. There is no statistical significance for a difference observed between 2 samples. There is no magic to RNA-Seq. These ideas are widely accepted for DNA microarray experiments, where a large number of biological replicates are now required to justify scientific conclusions . Hansen KD, Wu Z, Irizarry RA, Leek JT.  2011 Sequencing technology does not eliminate biological variability. Nat Biotechnol 29:572–573. [ abstract ] Reg. Experimental Design Sample Replicates for Expression Studies

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Key Advantages of RNA-Seq Provides a comprehensive view of the transcriptome. All transcripts can be analyzed (mRNA, ncRNA, snoRNA, lncRNA , miRNA, ...). Not necessarily dependent on any prior sequence knowledge. Increased dynamic range and tunable sensitivity . Can detect structural variations such as gene fusions and alternative splicing events. A truly digital solution (absolute abundance vs relative abundance). Microarray vs RNA Sequencing Key Advantages of Microarray Robust, reliable method, proven over decades of use High through-put method – 100s of samples analyzed per month Streamlined handling – can be easily automated Straightforward data analysis Short turn-around time – 5 days Lower cost

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Microfluidic Array Platform μ Paraflo ® Microfluidics Chip 10 µl total volume 4000 features 270 pl / reaction chamber uniform flow rate

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miRBase Version # of pre-cursor sequences (all species) Version 19 Aug 2012 2003 2011 Flexibility Allows miRBase Synchronicity

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Comprehensive Microarray Services Sample QC Sample preparation Hybridization reactions Advanced data analysis High level customer support Customer Total RNA Small RNA Isolation Labeling Customer Sequences Chip Design Chip Synthesis Chip QC Hybridization Signal Amplification Image Acquisition Customer Analysis Report Data Extraction Data Analysis Sample QC miRBase microRNA Microarray Expression Profiling

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Control / Treated Biological repeats t-Test p < 0.05 p < 0.01 Control Treated Multi-array normalization and clustering analysis Array assay Differentiated miRNAs of Biological & Statistical Significance - Multiple Chips microRNA Microarray Expression Profiling

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Repeats microRNA Microarray Expression Profiling

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Advanced Data Analysis Package Includes: The original and processed chip images An array layout file A raw intensity data file in Excel A fully analyzed data file in Excel A Data Summary containing a catalog of data files, images Images of representative regions of corresponding arrays Descriptions of specific features of the arrays A list of up and down regulated transcripts that are called based on a statistical analysis. microRNA Microarray Expression Profiling

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Key Advantages of RNA-Seq Provides a comprehensive view of the transcriptome. All transcripts can be analyzed (mRNA, ncRNA, snoRNA, lncRNA , miRNA, ...). Not necessarily dependent on any prior sequence knowledge. Increased dynamic range and tunable sensitivity . Can detect structural variations such as gene fusions and alternative splicing events. A truly digital solution (absolute abundance vs relative abundance). Microarray vs RNA Sequencing Key Advantages of Microarray Robust, reliable method, proven over decades of use High through-put method – 100s of samples analyzed per month Streamlined handling – can be easily automated Straightforward data analysis Short turn-around time – 5 days Lower cost

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Small RNA Sequencing and Data Analysis LC Sciences - Comprehensive RNA Sequencing Services Sample QC Sample preparation Library preparation High-throughput sequencing Advanced bioinformatics analysis High level customer support

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Sequencing Run Instrument: Illumina Genome Analyzer GAIIx Length of Reads: 35 bases Number of Reads: ~20-30 Million Data Output: ~0.7-1.0 Gb Bar-coding (Indexing) Samples: We recommend 3 per lane, Max 4 per lane The total number of reads does not change with bar-coding Sacrifice sequencing depth for lower cost Total Reads / Lane Number of Samples / Lane Reads/ Sample 30 M 1 30 M 30 M 2 15 M 30 M 3 10 M 30 M 4 7.5 M 30 M 5 6 M 30 M 6 5 M Small RNA Sequencing and Data Analysis

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Data Flow Mappable reads Raw reads Reads mapped to mammalian mirs in miRBase Reads un-mapped to mammalian mirs in miRBase mirs mapped to species genome mirs un-mapped to species genome Reads mapped to species genome Reads un-mapped to species genome Reads un-mapped to mRNA, Rfam, and repbase Reads mapped to mRNA, Rfam, and repbase Reads mapped to species genome Reads un-mapped to species genome Group 4 no hit others Group 1 Group 2 Group 3 Known species miRNAs Known miRNAs candidate species miRNAs Candidate species miRNAs genome inconsistent with miRBase Potentially novel miRNAs ACGT101-miR v3.5 Software Small RNA Sequencing and Data Analysis

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19,842,938 reads are mappable ADT, Junk, & Seq Filter 11,426,638 reads are mapped to miRBase or are miRNA candidates 2,400,521 reads mapped to mRNA, Rfam and repbase 6,015,779 reads - no hit 10,713,874 reads are filtered out 30,556,812 raw reads from sequencer Grp 1 - 8,007,998 Grp 2 - 14,067 Grp 3 - 64,086 Grp 4 - 3,340,487 64.9% 19.7% 15.1% 0.3% 57.6% 30.3% 12.1% 70.1% 29.2% 0.1% 0.6% Data Flow Small RNA Sequencing and Data Analysis

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Length Distribution of Mappable Reads Small RNA Sequencing and Data Analysis

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Can explain discrepancies array data vs qRT-PCR validation IsomiRs Small RNA Sequencing and Data Analysis

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Basic Research Specific miRNA up-regulation was monitored in stressed human primary neural (HNG) cells (a co-culture of neurons and astroglia). The inducible miRNA-125b and miRNA-146a, and their verified mRNA targets, including 15-lipoxygenase (15-LOX), synapsin-2 (SYN-2), complement factor H (CFH) and tetraspanin-12 (TSPAN12), suggests complex and highly interactive roles for NF- к B, miRNA-125b and miRNA-146a. These data further indicate that just two NF- к B-mediated miRNAs have tremendous potential to contribute to the regulation of neurotrophic support, synaptogenesis, neuroinflammation, innate immune signaling and amyloidogenesis in stressed primary neural cells of the human brain. Lukiw WJ. (2011) NF- к B-regulated micro RNAs (miRNAs) in primary human brain cells . Exp Neurol 235(2), 484-90. [ abstract ] NF-кB-regulated micro RNAs (miRNAs) in primary human brain cells Fig 2 - (A) Cluster diagram of NF- к B-up-regulated miRNAs in A β42 + IL-1 β- stressed HNG primary cells (N = 3) compared to untreated controls (N = 3); hsa miR = homo sapiens micro RNA; ; (B) dot blot confirmation of up-regulated miRNA-9, miRNA-125b and miRNA-146a abundance in stressed HNG cells compared to control 5SRNA and miRNA-183 (N = 2 control; N = 2 A β42 + IL-1 β- stressed)

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Molecular Diagnostics / Biomarkers in Body Fluids – Cerebral Spinal Fluid Baraniskin et al. (2011) Identification of microRNAs in the cerebrospinal fluid as marker for primary diffuse large B-cell lymphoma of the central nervous system . Blood 17(11):3140-6. [ article ] The authors demonstrate that miRNAs are present in the CSF of patients with PCNSL. With a candidate approach and miRNA quantification by reverse transcription polymerase chain reaction, miRNAs with significant levels in the CSF of patients with PCNSL were identified. More importantly, combined miRNA analyses resulted in an increased diagnostic accuracy with 95.7% sensitivity and 96.7% specificity. The study also demonstrated a remarkable stability of miRNAs in the CSF. CSF miRNA expression classification tree correctly diagnosing 95.7% of patients with PCNSL and 96.7% of control patients. Relative expression cutoff levels of ≥ 8.0 REL for miR-21, ≥ 1.4 REL for miR-19b, and ≥ 2.5 REL for miR-92a, respectively, were applied for diagnostic placements as depicted. CSF miRNAs are potentially useful tools as novel noninvasive biomarker for the diagnosis of PCNSL (Primary CNS Lymphoma)

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Molecular Diagnostics / Biomarkers in Body Fluids – Plasma Redell JB, Moore AN, Ward NH, Hergenroeder GW, Dash PK. (2010) Human Traumatic Brain Injury Alters Plasma microRNA Levels . J Neurotrauma 27(12), 2147-56. [ abstract ] Study examined if plasma miRNA levels are altered in patients with traumatic brain injury (TBI) relative to matched healthy volunteers, and explored their potential for use as diagnostic TBI biomarkers. The plasma miRNA profiles from severe TBI patients (Glasgow Coma Scale [GCS] score ≤8) and age-, gender-, and race-matched healthy volunteers were compared by microarray analysis. Of the 108 miRNAs identified in healthy volunteer plasma, 52 were altered after severe TBI, including 33 with decreased and 19 with increased relative abundance. The results presented herein indicate that circulating miRNAs hold promise as molecular biomarkers of TBI. Circulating miRNAs hold promise as molecular biomarkers of Traumatic Brain Injury Temporal changes in plasma miRNA content after traumatic brain injury. Purified RNA from plasma collected at various time points after injury were assayed using miRNA-specific TaqMan probes directed against (A) miR-16, (B) miR-26a, (C) miR-92a, (D) miR-638, or (E) miR-765. Changes in threshold cycle are relative to the mean of the healthy volunteer (HV) values. Data are presented as the mean ± standard error of the mean of each group (*p < 0.05 by one-way ANOVA, or ANOVA on ranks; ANOVA, analysis of variance; miRNA, microRNA; TBI, traumatic brain injury).

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Biomarkers – Molecular Diagnostics – Prognosis Teplyuk et al. (2012) MicroRNAs in cerebrospinal fluid identify glioblastoma and metastatic brain cancers and reflect disease activity . Neuro Oncol 14(6), 689-700. [ abstract ] Tested whether microRNA profiling of cerebrospinal fluid (CSF) enables detection of glioblastoma, discrimination between glioblastoma and metastatic brain tumors, and reflects disease activity. The pilot longitudinal study results indicate that a CSF miRNA profiling–based technique might be developed for disease monitoring and management ( eg , assessment of relapses and remissions, after treatment follow-up, and examination of chemo- and radiotherapy efficacy) which can help determine the most effective strategy of treatment. (A) Classification tree of brain cancer patients based on CSF miRNA biomarkers (miR-10b, -21, and -200). (B and C) Application of SVM with linear kernel to classification of specimens: (B) classification of GBM vs metastatic brain cancer on the basis of Ct levels of 2 miRNAs (miR-200a and miR-125b) in the CSF. (C) Classification of GBM and brain metastasis versus nonneoplastic control cases on the basis of Ct levels of 3 miRNAs (miR-10b, miR-200a, and miR-125b) in the CSF. miRNA profiling of CSF allows detection of GBM and metastatic brain cancers and miRNAs in CSF can serve as biomarkers of treatment response in brain cancers

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Drug Discovery / Therapeutics miRNAs as Drug Targets Specific miRNA abundance is significantly altered in neurological disorders such as Alzheimer disease (AD) when compared with age-matched controls. This study provides evidence in AD brains of a specific up-regulation of an NF-kappaB-sensitive miRNA-146a highly complementary to the 3'-untranslated region of complement factor H (CFH), an important repressor of the inflammatory response of the brain. These data indicate that NF-kappaB-sensitive miRNA-146a-mediated modulation of CFH gene expression may in part regulate an inflammatory response in AD brain and in stressed HN cell models of AD and illustrate the potential for anti-miRNAs as an effective therapeutic strategy against pathogenic inflammatory signaling. Fig 1 - miRNA-146a up-regulation in AD brain. A, merge of cy3/cy5 signals from a human micro-RNA panel (LC Sciences) indicates specific up-regulation of miRNA-146a (position A6; arrow). Levels of miRNA-9 (position A4), miRNA-132 (position A9), or miRNA-185 (position B6) showed no such changes. B, representative Northern hybridization of select brain-enriched miRNAs reconfirmed up-regulation of miR-146a (arrows). Position A1, miRNA-9; A2, miRNA-132; B1, miRNA-146a; B2, 5SRNA. An NF-B-sensitive miRNA-146a-mediated inflammatory circuit in Alzheimer Disease and in stressed Human brain cells Lukiw WJ, Zhao Y, Cui JG. (2008) An NF-kappaB-sensitive micro RNA-146a-mediated inflammatory circuit in Alzheimer disease and in stressed human brain cells. J Biol Chem 283(46), 31315-22. [ article ]

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Drug Discovery / Therapeutics miRNAs as Therapeutic Agents Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons, denervation of target muscles, muscle atrophy, and paralysis. Study shows that a key regulator of signaling between motor neurons and skeletal muscle fibers at neuromuscular synapses is miR-206, a skeletal muscle–specific microRNA that is dramatically induced in a mouse model of ALS. Deficiency of miR-206 in the ALS mouse model accelerates disease progression. Interventions to augment the sprouting of neuron terminals and their innervation of muscle might include small-molecule mimetics of miR-206 or perhaps miR-206 itself, agents that block repression of FGFBP1 by HDAC4, or inhibitors of HDAC4. MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice Williams AH, Valdez G, Moresi V, Qi X, McAnally J, Elliott JL, Bassel-Duby R, Sanes JR, Olson EN. (2009) MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice . Science 326(5959), 1549-554. [ abstract ]

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Society For Neuroscience Annual Meeting Booth #1917

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