DOCSLIB.ORG

Comparison of Microrna Deep Sequencing of Matched Formalin-Fixed Paraffin-Embedded and Fresh Frozen Cancer Tissues

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Comparison of Microrna Deep Sequencing of Matched Formalin-Fixed Paraffin-Embedded and Fresh Frozen Cancer Tissues Comparison of MicroRNA Deep Sequencing of Matched Formalin-Fixed Paraffin-Embedded and Fresh Frozen Cancer Tissues Wei Meng1, Joseph P. McElroy2, Stefano Volinia3, Jeff Palatini4, Sarah Warner4, Leona W. Ayers5, Kamalakannan Palanichamy1, Arnab Chakravarti1*, Tim Lautenschlaeger1* 1 Department of Radiation Oncology, Wexner Medical Center, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University, Columbus, Ohio, United States of America, 2 Center for Biostatistics, The Ohio State University, Columbus, Ohio, United States of America, 3 Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio, United States of America, 4 Microarray Shared Resource, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University, Columbus, Ohio, United States of America, 5 Department of Pathology, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America Abstract MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin- fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (2–9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t- test p.0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson’s r = 0.71–0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified. Citation: Meng W, McElroy JP, Volinia S, Palatini J, Warner S, et al. (2013) Comparison of MicroRNA Deep Sequencing of Matched Formalin-Fixed Paraffin- Embedded and Fresh Frozen Cancer Tissues. PLoS ONE 8(5): e64393. doi:10.1371/journal.pone.0064393 Editor: Soheil S. Dadras, University of Connecticut Health Center, United States of America Received January 2, 2013; Accepted April 13, 2013; Published May 16, 2013 Copyright: ß 2013 Meng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Department of Radiation Oncology and the Comprehensive Cancer Center, and, by the Biostatistics Core and by Microarray shared resource at The Ohio State University. The work was supported by Award Number Grant 8UL1TR000090-05 from the National Center For Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Advancing Translational Sciences or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (AC); [email protected] (TL) Introduction formalin-fixation and paraffin-embedding (FFPE). Most clinical assays performed in pathology laboratories are optimized for MicroRNAs are noncoding small RNAs with a length of 22 to FFPE tissues. FFPE tissues are typically stored at room temper- 26 nucleotides (nt). MicroRNAs result in degradation of comple- ature. Formalin preserves tissue samples by creating cross-linking mentary messenger RNA in many species. MicroRNAs play an between proteins, DNA and RNA. DNA extracted from FFPE important role in cell development, cell death, cell proliferation tissue has been shown to be useful for copy number analysis and [1], apoptosis [2] and angiogenesis [3]. The pioneer bead-based mutation analysis on at least one platform in some settings [12]. flow cytometric microRNA expression profiling method revealed However, the chemical modification between macromolecules and that microRNA profiles reflect the developmental lineage and RNAs caused by formalin fixation can accelerate the degradation differentiation of tumors [4]. Normal tissues and cancer tissues of RNA [13] [14]. The stability of microRNAs is generally much were shown to have distinctive expression profiles and microRNA more robust than messenger RNA [15]. Next generation profiles can describe microRNA-driven pathways in solid tumors sequencing based microRNA expression analysis has been [5]. developed on several platforms, including Roche/454 platform, The current methods for microRNA quantification are: real- Illumina’s Genome Analyzer and ABI’s SOLiD platform [16] time reverse transcription-PCR [6,7], microarray [8], Nanostring, [17], and different commercial protocols for miRNA library and next generation sequencing [9–11]. Especially, the newly preparation have been developed by the three companies [18]. developed next generation sequencing technologies have the There is currently only limited evidence available that microRNA potential to discover novel microRNAs and other small RNAs. sequencing results are reliable and valid. Ma et al. demonstrated Most tissues processed in the health care setting will undergo the feasibility of miRNA profiling based on Sanger sequencing in PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e64393 miRNA Deep Sequencing in Cancer Tissues 10-year-old archived tissue [19]. To our knowledge there are only RNA Isolation, quantitation, and quality Assessment two peer-reviewed studies published that compared microRNA RNA from FFPE samples were extracted using Recover All sequencing results of matched frozen and FFPE specimens using Total Nucleic Acid Isolation Kit (Life Technologies Corporation, the Illumina platform [9,20]. The objective of this study was to Carlsbad, CA). Briefly, 5–10 mg samples were sliced from paraffin determine if FFPE samples can be successfully characterized by blocks and deparaffinizated by xylene at 50uC, followed by 100% deep miRNA sequencing. To this end we analyzed matched ethanol wash. The air-dry tissue samples were digested by frozen and FFPE tissues of different histologies for which detailed proteinase K for 24 hrs in a microtube shaking incubator set at processing and storage information was available. 50uC. The digested samples were mixed with appropriate volume of isolation additive and 100% ethanol. After passing the mixture Materials and Methods through the filter cartridge, the DNA and RNA were retained on the filter. The DNA was removed by on-filter DNase digestion. Ethics Statement The RNA was purified by washing buffer and eluted with Human malignant tissue samples procured during period 2003– nuclease-free water. 2009 were obtained from the Cooperative Human Tissue Network The microRNA samples from the fresh frozen samples were (CHTN/NCI), Midwestern Division, which is funded by the extracted using PureLinkTM miRNA Isolation Kit (Life Technol- National Cancer Institute, based on an internal review board ogies Corporation, Carlsbad, CA). Briefly, 5 mg fresh frozen tissue (IRB) approved research protocol (Ohio State University Human samples were mixed with 300 ml Binding Buffer (L3) and Subjects Protocol - 2011E0377). Other Investigators may have homogenized using tissue homogenizer. The homogenized sam- received specimens from the same subjects. ples were centrifuged, and supernatant were mixed with 300 ml 70% ethanol. The solution containing the RNA was purified by Tissue specimens two round of spin columns cleanup. The RNA was eluted with 50– Eight paired human malignant tissue samples were received. 100 ml sterile RNase-free water. Remnant surgical tissues were taken after diagnostic samples were secured from patients (five males and four females; median age 58 MicroRNA sequencing years, range 39–78 years) with breast invasive ductal carcinoma Small RNAs from FFPE and fresh frozen samples were prepared (2), renal clear cell carcinoma (2), lung adenocarcinoma (1), for SOLiD sequencing as follows: the total RNA samples were prostate adenocarcinoma (1), metastatic melanoma (1), and processed by the flashPAGE fractionator (Ambion) and flashPAGE sarcoma of thigh (1). Surgical specimens were transported from Clean-Up Kit (Ambion). The enriched small RNA was then the operating rooms to Tissue Procurement, examined under a processed according to the SOLiD Small RNA Expression Kit pathologist’s supervision and preserved within 5 to 57 recorded protocol (Applied Biosystems). The purified small RNAs were minutes. Paired tissue samples selected for research were either ligated with 5’ and 3’ adapter mix using RNA ligase. The ligated immediately snap frozen in liquid nitrogen and placed in a –80uC products (40–60 bases in length) were reverse transcribed and freezer for monitored storage
Load more

Recommended publications
  • Next Generation DNA Sequencing
    Genes 2010, 1, 385-387; doi:10.3390/genes1030385 OPEN ACCESS genes ISSN 2073-4425 www.mdpi.com/journal/genes Editorial Special Issue: Next Generation DNA Sequencing Paul Richardson PRC LLC, 25 Amber Lane, Lafayette, CA 94549, USA; E-Mail: [email protected]; Tel.: +1 925 301-5460 Received: 21 October 2010 / Accepted: 26 October 2010 / Published: 27 October 2010 Next Generation Sequencing (NGS) refers to technologies that do not rely on traditional dideoxy-nucleotide (Sanger) sequencing where labeled DNA fragments are physically resolved by electrophoresis. These new technologies rely on different strategies, but essentially all of them make use of real-time data collection of a base level incorporation event across a massive number of reactions (on the order of millions versus 96 for capillary electrophoresis for instance). The major commercial NGS platforms available to researchers are the 454 Genome Sequencer (Roche), Illumina (formerly Solexa) Genome analyzer, the SOLiD system (Applied Biosystems/Life Technologies) and the Heliscope (Helicos Corporation). The techniques and different strategies utilized by these platforms are reviewed in a number of the papers in this special issue. These technologies are enabling new applications that take advantage of the massive data produced by this next generation of sequencing instruments. In this special issue, nine papers review and demonstrate the utility and potential of next generation sequencing. One of the biggest consequences with NGS technologies is how to deal with all of the data produced by these platforms. Magi et al. [1] review the software tools available for the multiple functions needed to process and interpret the huge amounts of data produced by these instruments.
    [Show full text]
  • Evaluation of Normalization Methods in Mammalian Microrna-Seq Data
    Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Evaluation of normalization methods in mammalian microRNA-Seq data LANA XIA GARMIRE1 and SHANKAR SUBRAMANIAM1 Department of Bioengineering, Jacobs School of Engineering, University of California at San Diego, La Jolla, California 92093-0412, USA ABSTRACT Simple total tag count normalization is inadequate for microRNA sequencing data generated from the next generation sequencing technology. However, so far systematic evaluation of normalization methods on microRNA sequencing data is lacking. We comprehensively evaluate seven commonly used normalization methods including global normalization, Lowess normalization, Trimmed Mean Method (TMM), quantile normalization, scaling normalization, variance stabilization, and invariant method. We assess these methods on two individual experimental data sets with the empirical statistical metrics of mean square error (MSE) and Kolmogorov-Smirnov (K-S) statistic. Additionally, we evaluate the methods with results from quantitative PCR validation. Our results consistently show that Lowess normalization and quantile normalization perform the best, whereas TMM, a method applied to the RNA-Sequencing normalization, performs the worst. The poor performance of TMM normalization is further evidenced by abnormal results from the test of differential expression (DE) of microRNA-Seq data. Comparing with the models used for DE, the choice of normalization method is the primary factor that affects the results of DE. In summary, Lowess normalization and quantile normalization are recommended for normalizing microRNA-Seq data, whereas the TMM method should be used with caution. Keywords: microRNA-Seq; next generation sequencing; statistical normalization; high-throughput data analysis INTRODUCTION much smaller than mRNAs. For example, so far there are <1000 annotated microRNAs in human that are expected to The next generation sequencing (NGS) technology has regulate z30% of genes.
    [Show full text]
  • Ancient Microrna Profiles of a 14,300-Year-Old Canid Samples Confirm
    Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al 1 2 Title: Ancient microRNA profiles of a 14,300-year-old canid samples confirm 3 taxonomic origin and give glimpses into tissue-specific gene regulation from 4 the Pleistocene 5 6 Running title: Sequencing of ancient microRNAs. 7 8 Authors: Bastian Fromm1*, Marcel Tarbier1, Oliver Smith2,3, Emilio Mármol- 9 Sánchez1,4, Love Dalén4,5, M. Tom P. Gilbert2,6, Marc R. Friedländer1* 10 11 Affiliations: 12 1 Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren 13 Institute, Stockholm University, Stockholm, Sweden 14 2 Section for Evolutionary Genomics, The Globe Institute, Faculty of Health and Medical 15 Sciences, University of Copenhagen, Copenhagen, Denmark 16 3 Micropathology Ltd, Venture Centre, University of Warwick Science Park, Coventry, UK 17 4 Centre for Palaeogenetics, Stockholm, Sweden 18 5 Department of Bioinformatics and Genetics, Swedish Museum of Natural History, 19 SE-10405 Stockholm, Sweden 20 6 Norwegian University of Science and Technology, University Museum, Trondheim, 21 Norway 22 23 24 * corresponding authors: [email protected] and [email protected] 25 26 Keywords: microRNAs, paleotranscriptomics, Pleistocene, canid, gene regulation Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Fromm et al. 27 Abstract 28 DNA sequencing is the current key technology for historic or ancient biological samples and has 29 led to many exciting discoveries in the field of paleogenomics. However, functional insights into 30 tissue identity, cellular composition or gene regulation cannot be gained from DNA.
    [Show full text]
  • Downregulation of Hsa-Microrna-204-5P and Identification of Its Potential Regulatory Network in Non-Small Cell Lung Cancer: RT-Q
    Liang et al. Respiratory Research (2020) 21:60 https://doi.org/10.1186/s12931-020-1274-9 RESEARCH Open Access Downregulation of hsa-microRNA-204-5p and identification of its potential regulatory network in non-small cell lung cancer: RT- qPCR, bioinformatic- and meta-analyses Chang-Yu Liang1†, Zu-Yun Li1†, Ting-Qing Gan2†, Ye-Ying Fang3†, Bin-Liang Gan1†, Wen-Jie Chen1†, Yi-Wu Dang1†, Ke Shi1†, Zhen-Bo Feng1* and Gang Chen1* Abstract Background: Pulmonary malignant neoplasms have a high worldwide morbidity and mortality, so the study of these malignancies using microRNAs (miRNAs) has attracted great interest and enthusiasm. The aim of this study was to determine the clinical effect of hsa-microRNA-204-5p (miR-204-5p) and its underlying molecular mechanisms in non-small cell lung cancer (NSCLC). Methods: Expression of miR-204-5p was investigated by real-time quantitative PCR (RT-qPCR). After data mining from public online repositories, several integrative assessment methods, including receiver operating characteristic (ROC) curves, hazard ratios (HR) with 95% confidence intervals (95% CI), and comprehensive meta-analyses, were conducted to explore the expression and clinical utility of miR-204-5p. The potential objects regulated and controlled by miR-204-5p in the course of NSCLC were identified by estimated target prediction and analysis. The regulatory network of miR-204-5p, with its target genes and transcription factors (TFs), was structured from database evidence and literature references. Results: The expression of miR-204-5p was downregulated in NSCLC, and the downtrend was related to gender, histological type, vascular invasion, tumor size, clinicopathologic grade and lymph node metastasis (P<0.05).
    [Show full text]
  • Analyzing Mirna from Raw NGS Data to Quantification
    G C A T T A C G G C A T genes Article DIANA-mAP: Analyzing miRNA from Raw NGS Data to Quantification Athanasios Alexiou 1,2, Dimitrios Zisis 2, Ioannis Kavakiotis 1, Marios Miliotis 1,2, Antonis Koussounadis 3 , Dimitra Karagkouni 1,2 and Artemis G. Hatzigeorgiou 1,2,3,* 1 DIANA Lab, Department of Computer Science and Biomedical Informatics, University of Thessaly, 35131 Lamia, Greece; [email protected] (A.A.); [email protected] (I.K.); [email protected] (M.M.); [email protected] (D.K.) 2 Hellenic Pasteur Institute, 11521 Athens, Greece; [email protected] 3 Department of Electrical & Computer Engineering, University of Thessaly, 38221 Volos, Greece; [email protected] * Correspondence: [email protected]; Tel.: +30-24210-74758; Fax: +30-24210-74997 Abstract: microRNAs (miRNAs) are small non-coding RNAs (~22 nts) that are considered central post-transcriptional regulators of gene expression and key components in many pathological con- ditions. Next-Generation Sequencing (NGS) technologies have led to inexpensive, massive data production, revolutionizing every research aspect in the fields of biology and medicine. Particularly, small RNA-Seq (sRNA-Seq) enables small non-coding RNA quantification on a high-throughput scale, providing a closer look into the expression profiles of these crucial regulators within the cell. Here, we present DIANA-microRNA-Analysis-Pipeline (DIANA-mAP), a fully automated compu- tational pipeline that allows the user to perform miRNA NGS data analysis from raw sRNA-Seq libraries to quantification and Differential Expression Analysis in an easy, scalable, efficient, and intuitive way. Emphasis has been given to data pre-processing, an early, critical step in the anal- ysis for the robustness of the final results and conclusions.
    [Show full text]
  • Addressing Bias in Small RNA Library Preparation for Sequencing: a New Protocol Recovers Micrornas That Evade Capture by Current Methods
    ORIGINAL RESEARCH published: 22 December 2015 doi: 10.3389/fgene.2015.00352 Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods Jeanette Baran-Gale1,2*,C.LisaKurtz2, Michael R. Erdos3, Christina Sison4, Alice Young4, Emily E. Fannin2, Peter S. Chines3 and Praveen Sethupathy1,2* 1 Bioinformatics and Computational Biology Curriculum, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2 Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 3 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA, 4 NIH Intramural Sequencing Center, National Institutes of Health, Rockville, MD, USA Edited by: Ghanshyam Upadhyay, Recent advances in sequencing technology have helped unveil the unexpected City College of New York-CUNY, USA Reviewed by: complexity and diversity of small RNAs. A critical step in small RNA library preparation Sandeep Kumar, for sequencing is the ligation of adapter sequences to both the 5 and 3 ends of small State University of New York College RNAs. Studies have shown that adapter ligation introduces a significant but widely of Optometry, USA Ravi C. Kalathur, unappreciated bias in the results of high-throughput small RNA sequencing. We show New York Structural Biology Center, that due to this bias the two widely used Illumina library preparation protocols produce USA strikingly different microRNA (miRNA) expression profiles in the same batch of cells. Manish Mishra, All Saints University College There are 102 highly expressed miRNAs that are >5-fold differentially detected and of Medicine, Saint Vincent some miRNAs, such as miR-24-3p, are over 30-fold differentially detected.
    [Show full text]
  • Microrna and Circrna Expression Analysis in a Zbtb1 Gene Knockout Monoclonal EL4 Cell Line
    ORIGINAL RESEARCH published: 05 July 2021 doi: 10.3389/fcimb.2021.706919 MicroRNA and circRNA Expression Analysis in a Zbtb1 Gene Knockout Monoclonal EL4 Cell Line † † Jun-Hong Wang 1,2,3 , Chun-Wei Shi 1,2,3 , Yi-Yuan Lu 1,2,3, Yan Zeng 1,2,3, Ming-Yang Cheng 1,2,3, Ru-Yu Wang 1,2,3,YuSun 1,2,3, Yan-Long Jiang 1,2,3, Wen-Tao Yang 1,2,3, Dan-Dan Zhao 1,2,3, Hai-Bin Huang 1,2,3, Li-Ping Ye 1,2,3, Xin Cao 1,2,3*, Gui-Lian Yang 1,2,3* and Chun-Feng Wang 1,2,3* 1 College of Veterinary Medicine, Jilin Agricultural University, Changchun, China, 2 Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, Changchun, China, 3 Key Laboratory of Animal Production and Product Quality Safety of Ministry of Education, Jilin Agricultural University, Changchun, China Edited by: Yongsheng Liu, Chinese Academy of Agricultural Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, Sciences, China and a member of the mammalian Zbtb gene family. Previous studies have shown that Reviewed by: Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is Peng Tan, Broad Institute, United States undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the Dongqin Yang, CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by Fudan University, China the control and gene deletion groups were determined by RNA-seq.
    [Show full text]
  • Single-Cell Microrna Sequencing Method Comparison and Application to Cell Lines and Circulating Lung Tumor Cells
    ARTICLE https://doi.org/10.1038/s41467-021-24611-w OPEN Single-cell microRNA sequencing method comparison and application to cell lines and circulating lung tumor cells Sarah M. Hücker 1,8, Tobias Fehlmann 2,8, Christian Werno1, Kathrin Weidele1, Florian Lüke1,3, ✉ Anke Schlenska-Lange4, Christoph A. Klein 1,5, Andreas Keller 2,6,7,9 & Stefan Kirsch 1,9 1234567890():,; Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1 pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best protocols. Third, we sequence single cells from eight different cell lines and 67 circulating tumor cells (CTCs) from seven SCLC patients. Altogether, we analyze 244 different samples. We observe high reproducibility within pro- tocols and reads covered a broad spectrum of RNAs. For the 67 CTCs, we detect a median of 68 miRNAs, with 10 miRNAs being expressed in 90% of tested cells. Enrichment analysis suggested the lung as the most likely organ of origin and enrichment of cancer-related categories. Even the identification of non-annotated candidate miRNAs was feasible, underlining the potential of single cell small RNA sequencing. 1 Division of Personalized Tumor Therapy, Fraunhofer-Institute for Toxicology and Experimental Medicine, Regensburg, Germany. 2 Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany. 3 Department of Internal Medicine III Hematology & Oncology, University Medical Center Regensburg, Regensburg, Germany. 4 Department of Oncology and Hematology, Hospital Barmherzige Brüder, Regensburg, Germany.
    [Show full text]
  • Erythrocyte Microrna Sequencing Reveals Differential Expression in Relapsing-Remitting Multiple Sclerosis Kira Groen1,2, Vicki E
    Groen et al. BMC Medical Genomics (2018) 11:48 https://doi.org/10.1186/s12920-018-0365-7 RESEARCH ARTICLE Open Access Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis Kira Groen1,2, Vicki E. Maltby1,2, Rodney A. Lea2,3, Katherine A. Sanders4, J. Lynn Fink5, Rodney J. Scott2,6,7, Lotti Tajouri8 and Jeannette Lechner-Scott1,2,9* Abstract Background: There is a paucity of knowledge concerning erythrocytes in the aetiology of Multiple Sclerosis (MS) despite their potential to contribute to disease through impaired antioxidant capacity and altered haemorheological features. Several studies have identified an abundance of erythrocyte miRNAs and variable profiles associated with disease states, such as sickle cell disease and malaria. The aim of this study was to compare the erythrocyte miRNA profile of relapsing-remitting MS (RRMS) patients to healthy sex- and age-matched controls. Methods: Erythrocytes were purified by density-gradient centrifugation and RNA was extracted. Following library preparation, samples were run on a HiSeq4000 Illumina instrument (paired-end 100 bp sequencing). Sequenced erythrocyte miRNA profiles (9 patients and 9 controls) were analysed by DESeq2. Differentially expressed miRNAs were validated by RT-qPCR using miR-152-3p as an endogenous control and replicated in a larger cohort (20 patients and 18 controls). After logarithmic transformation, differential expression was determined by two-tailed unpaired t-tests. Logistic regression analysis was carried out and receiver operating characteristic (ROC) curves were generated to determine biomarker potential. Results: A total of 236 erythrocyte miRNAs were identified. Of twelve differentially expressed miRNAs in RRMS two showed increased expression (adj.
    [Show full text]
  • Computational Analysis of Microrna Function
    microRNA and smallRNA profiling Anton Enright Group Leader EMBL - European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK http://www.ebi.ac.uk/enright/ [email protected] High Throughput Sequencing Analysis 2013 Thursday, 24 October 13 microRNA Regulation Adapted from He L. and Hannon G.J; Nature reviews genetics 5:552 (2004) EMBL-EBI 2 High throughput RNA Seq Thursday, 24 October 13 microRNA Regulation Adapted from He L. and Hannon G.J; Nature reviews genetics 5:552 (2004) EMBL-EBI 2 High throughput RNA Seq Thursday, 24 October 13 MicroRNA Processing EMBL-EBI 3 High throughput RNA Seq Thursday, 24 October 13 MicroRNA Processing EMBL-EBI 3 High throughput RNA Seq Thursday, 24 October 13 MicroRNA Processing EMBL-EBI 3 High throughput RNA Seq Thursday, 24 October 13 microRNA Profiling EMBL-EBI 4 High throughput RNA Seq Thursday, 24 October 13 microRNA Profiling • MicroRNA microarray • Statistical issues with number of probes • Cross-hybridisation and biases • Cheap and straightforward • Small RNA Sequencing • Quantitation can be difficult due to biases • Ability to detect novel microRNAs, edits and variation • Reproducible • qRT-PCR Based Approaches • quite accurate • lower throughput • Single Molecule profiling • e.g. Nanostring • New technology EMBL-EBI 5 High throughput RNA Seq Thursday, 24 October 13 Nanostring EMBL-EBI 6 High throughput RNA Seq Thursday, 24 October 13 Nanostring EMBL-EBI 6 High throughput RNA Seq Thursday, 24 October 13 microRNA microarrays • MicroRNA array manufacturers love to talk about how many probes they have on their chips. • In reality there are < 1000 microRNAs for your species and likely <30 expressed in your sample.
    [Show full text]
  • Deep-Sequencing of Endothelial Cells Exposed to Hypoxia Reveals the Complexity of Known and Novel Micrornas
    Downloaded from rnajournal.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs CHRISTINE VOELLENKLE,1,6 JEROEN VAN ROOIJ,1,6 ALESSANDRO GUFFANTI,2,6 ELENA BRINI,2 PASQUALE FASANARO,3 ELEONORA ISAIA,1 LARRY CROFT,4 MATEI DAVID,5 MAURIZIO C. CAPOGROSSI,3 ANNA MOLES,2 ARMANDO FELSANI,2 and FABIO MARTELLI3,7 1Laboratorio di Cardiologia Molecolare, IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy 2Genomnia srl, Lainate, 20020 Milan, Italy 3Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, 00167 Rome, Italy 4Institute for Molecular Bioscience, University of Queensland, 4072 Brisbane, Australia 5Computational Biology Laboratory, University of Toronto, M5S 3G4 Toronto, Canada ABSTRACT In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1%% O2 or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/microRNA* species with a broad abundance range: miR-21 and miR-126 totaled almost 40%% of all miRNAs. A complex repertoire of isomiRs was found, displaying also 59 variations, potentially affecting target recognition. High- stringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex.
    [Show full text]
  • Mirge 2.0 for Comprehensive Analysis of Microrna Sequencing Data Yin Lu, Alexander S
    Lu et al. BMC Bioinformatics (2018) 19:275 https://doi.org/10.1186/s12859-018-2287-y SOFTWARE Open Access miRge 2.0 for comprehensive analysis of microRNA sequencing data Yin Lu, Alexander S. Baras and Marc K. Halushka* Abstract Background: miRNAs play important roles in the regulation of gene expression. The rapidly developing field of microRNA sequencing (miRNA-seq; small RNA-seq) needs comprehensive, robust, user-friendly and standardized bioinformatics tools to analyze these large datasets. We present miRge 2.0, in which multiple enhancements were made towards these goals. Results: miRge 2.0 has become more comprehensive with increased functionality including a novel miRNA detection method, A-to-I editing analysis, integrated standardized GFF3 isomiR reporting, and improved alignment to miRNAs. The novel miRNA detection method uniquely uses both miRNA hairpin sequence structure and composition of isomiRs resulting in higher specificity for potential miRNA identification. Using known miRNA data, our support vector machine (SVM) model predicted miRNAs with an average Matthews correlation coefficient (MCC) of 0.939 over 32 human cell datasets and outperformed miRDeep2 and miRAnalyzer regarding phylogenetic conservation. The A-to-I editing detection strongly correlated with a reference dataset with adjusted R2 = 0.96. miRge 2.0 is the most up-to-date aligner with custom libraries to both miRBase v22 and MirGeneDB v2.0 for 6 species: human, mouse, rat, fruit fly, nematode and zebrafish; and has a tool to create custom libraries. For user-friendliness, miRge 2.0 is incorporated into bcbio- nextgen and implementable through Bioconda. Conclusions: miRge 2.0 is a redesigned, leading miRNA RNA-seq aligner with several improvements and novel utilities.
    [Show full text]