Live Imaging of Nascent RNA Dynamics Reveals Distinct Types of Transcriptional Pulse Regulation
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Ovulation-Selective Genes: the Generation and Characterization of an Ovulatory-Selective Cdna Library
531 Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library A Hourvitz1,2*, E Gershon2*, J D Hennebold1, S Elizur2, E Maman2, C Brendle1, E Y Adashi1 and N Dekel2 1Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA 2Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel (Requests for offprints should be addressed to N Dekel; Email: [email protected]) *(A Hourvitz and E Gershon contributed equally to this paper) (J D Hennebold is now at Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon 97006, USA) Abstract Ovulation-selective/specific genes, that is, genes prefer- (FAE-1) homolog, found to be localized to the inner entially or exclusively expressed during the ovulatory periantral granulosa and to the cumulus granulosa cells of process, have been the subject of growing interest. We antral follicles. The FAE-1 gene is a -ketoacyl-CoA report herein studies on the use of suppression subtractive synthase belonging to the fatty acid elongase (ELO) hybridization (SSH) to construct a ‘forward’ ovulation- family, which catalyzes the initial step of very long-chain selective/specific cDNA library. In toto, 485 clones were fatty acid synthesis. All in all, the present study accom- sequenced and analyzed for homology to known genes plished systematic identification of those hormonally with the basic local alignment tool (BLAST). Of those, regulated genes that are expressed in the ovary in an 252 were determined to be nonredundant. -
18S Rrna Is a Reliable Normalisation Gene for Real Time PCR Based On
Kuchipudi et al. Virology Journal 2012, 9:230 http://www.virologyj.com/content/9/1/230 METHODOLOGY Open Access 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells Suresh V Kuchipudi1*, Meenu Tellabati1, Rahul K Nelli1, Gavin A White2, Belinda Baquero Perez1, Sujith Sebastian1, Marek J Slomka3, Sharon M Brookes3, Ian H Brown3, Stephen P Dunham1 and Kin-Chow Chang1 Abstract Background: One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. Results: The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. -
Systematic Identification of Housekeeping Genes Possibly Used As References in Caenorhabditis Elegans by Large-Scale Data Integration
cells Article Systematic Identification of Housekeeping Genes Possibly Used as References in Caenorhabditis elegans by Large-Scale Data Integration 1, 1, 1 1 1 1 1 Jingxin Tao y, Youjin Hao y, Xudong Li , Huachun Yin , Xiner Nie , Jie Zhang , Boying Xu , Qiao Chen 2 and Bo Li 1,* 1 College of Life Sciences, Chongqing Normal University, Chongqing 401331, China; [email protected] (J.T.); [email protected] (Y.H.); [email protected] (X.L.); [email protected] (H.Y.); [email protected] (X.N.); [email protected] (J.Z.); [email protected] (B.X.) 2 Scientific Research Office, Chongqing Normal University, Chongqing 401331, China; [email protected] * Correspondence: [email protected]; Tel.: +86-23-6591-0315 These authors contributed equally to this work. y Received: 24 January 2020; Accepted: 11 March 2020; Published: 24 March 2020 Abstract: For accurate gene expression quantification, normalization of gene expression data against reliable reference genes is required. It is known that the expression levels of commonly used reference genes vary considerably under different experimental conditions, and therefore, their use for data normalization is limited. In this study, an unbiased identification of reference genes in Caenorhabditis elegans was performed based on 145 microarray datasets (2296 gene array samples) covering different developmental stages, different tissues, drug treatments, lifestyle, and various stresses. As a result, thirteen housekeeping genes (rps-23, rps-26, rps-27, rps-16, rps-2, rps-4, rps-17, rpl-24.1, rpl-27, rpl-33, rpl-36, rpl-35, and rpl-15) with enhanced stability were comprehensively identified by using six popular normalization algorithms and RankAggreg method. -
Analysis of the Stability of 70 Housekeeping Genes During Ips Reprogramming Yulia Panina1,2*, Arno Germond1 & Tomonobu M
www.nature.com/scientificreports OPEN Analysis of the stability of 70 housekeeping genes during iPS reprogramming Yulia Panina1,2*, Arno Germond1 & Tomonobu M. Watanabe1 Studies on induced pluripotent stem (iPS) cells highly rely on the investigation of their gene expression which requires normalization by housekeeping genes. Whether the housekeeping genes are stable during the iPS reprogramming, a transition of cell state known to be associated with profound changes, has been overlooked. In this study we analyzed the expression patterns of the most comprehensive list to date of housekeeping genes during iPS reprogramming of a mouse neural stem cell line N31. Our results show that housekeeping genes’ expression fuctuates signifcantly during the iPS reprogramming. Clustering analysis shows that ribosomal genes’ expression is rising, while the expression of cell-specifc genes, such as vimentin (Vim) or elastin (Eln), is decreasing. To ensure the robustness of the obtained data, we performed a correlative analysis of the genes. Overall, all 70 genes analyzed changed the expression more than two-fold during the reprogramming. The scale of this analysis, that takes into account 70 previously known and newly suggested genes, allowed us to choose the most stable of all genes. We highlight the fact of fuctuation of housekeeping genes during iPS reprogramming, and propose that, to ensure robustness of qPCR experiments in iPS cells, housekeeping genes should be used together in combination, and with a prior testing in a specifc line used in each study. We suggest that the longest splice variants of Rpl13a, Rplp1 and Rps18 can be used as a starting point for such initial testing as the most stable candidates. -
Transcriptional Burst Frequency and Burst Size Are Equally Modulated Across the Human Genome
Transcriptional burst frequency and burst size are equally modulated across the human genome Roy D. Dara,b,c,1, Brandon S. Razookya,d,e,1, Abhyudai Singhd,2, Thomas V. Trimelonif, James M. McCollumf, Chris D. Coxg,h, Michael L. Simpsonb,I,3, and Leor S. Weinbergera,d,j,3 aGladstone Institutes, San Francisco, CA 94158; bCenter for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831; cDepartment of Physics and Astronomy, University of Tennessee, Knoxville, TN 37996; dDepartment of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093; eBiophysics Graduate Group, University of California, San Francisco, CA 94158; gCenter for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996;hDepartment of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 37996; IDepartment of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996; jDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; and fDepartment of Electrical and Computer Engineering, Virginia Commonwealth University, Richmond, VA 23284–3072 Edited by Jonathan S. Weissman, University of California, San Francisco, CA, and approved September 13, 2012 (received for review August 8, 2012) Gene expression occurs either as an episodic process, characterized autocorrelation time of expression fluctuations (as measured by pulsatile bursts, or as a constitutive process, characterized by by the noise autocorrelation time at half of its initial value, τ1∕2) a Poisson-like accumulation of gene products. It is not clear which (11, 12) (Fig. 1C). Although this three-dimensional noise space is mode of gene expression (constitutive versus bursty) predomi- impractical to analyze directly, different two-dimensional projec- nates across a genome or how transcriptional dynamics are influ- tions of noise space allow the quantification of rate parameters enced by genomic position and promoter sequence. -
Genome-Wide Analysis of Transcriptional Bursting-Induced Noise in Mammalian Cells
bioRxiv preprint doi: https://doi.org/10.1101/736207; this version posted August 15, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Title: Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells Authors: Hiroshi Ochiai1*, Tetsutaro Hayashi2, Mana Umeda2, Mika Yoshimura2, Akihito Harada3, Yukiko Shimizu4, Kenta Nakano4, Noriko Saitoh5, Hiroshi Kimura6, Zhe Liu7, Takashi Yamamoto1, Tadashi Okamura4,8, Yasuyuki Ohkawa3, Itoshi Nikaido2,9* Affiliations: 1Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-0046, Japan 2Laboratory for Bioinformatics Research, RIKEN BDR, Wako, Saitama, 351-0198, Japan 3Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka, 812-0054, Japan 4Department of Animal Medicine, National Center for Global Health and Medicine (NCGM), Tokyo, 812-0054, Japan 5Division of Cancer Biology, The Cancer Institute of JFCR, Tokyo, 135-8550, Japan 6Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa, 226-8503, Japan 7Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA 8Section of Animal Models, Department of Infectious Diseases, National Center for Global Health and Medicine (NCGM), Tokyo, 812-0054, Japan 9Bioinformatics Course, Master’s/Doctoral Program in Life Science Innovation (T-LSI), School of Integrative and Global Majors (SIGMA), University of Tsukuba, Wako, 351-0198, Japan *Corresponding authors Corresponding authors e-mail addresses Hiroshi Ochiai, [email protected] Itoshi Nikaido, [email protected] bioRxiv preprint doi: https://doi.org/10.1101/736207; this version posted August 15, 2019. -
A Common Analgesic Enhances the Anti-Tumour Activity of 5-Aza-2’- Deoxycytidine Through Induction of Oxidative Stress
bioRxiv preprint doi: https://doi.org/10.1101/2020.03.31.017947; this version posted April 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A common analgesic enhances the anti-tumour activity of 5-aza-2’- deoxycytidine through induction of oxidative stress Hannah J. Gleneadie1,10, Amy H. Baker1, Nikolaos Batis2, Jennifer Bryant2, Yao Jiang3, Samuel J.H. Clokie4, Hisham Mehanna2, Paloma Garcia5, Deena M.A. Gendoo6, Sally Roberts5, Alfredo A. Molinolo7, J. Silvio Gutkind8, Ben A. Scheven1, Paul R. Cooper1, Farhat L. Khanim9 and Malgorzata Wiench1, 5,*. 1School of Dentistry, Institute of Clinical Studies, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B5 7EG, UK; 2Institute of Head and Neck Studies and Education (InHANSE), The University of Birmingham, Birmingham, B15 2TT, UK; 3School of Biosciences, The University of Birmingham, Birmingham, B15 2TT, UK; 4West Midlands Regional Genetics Laboratory, Birmingham Women’s and Children’s Hospital, Birmingham, B15 2TG, UK; 5Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, B15 2TT, UK; 6Centre for Computational Biology, Institute of Cancer and Genomic Sciences, The University of Birmingham, Birmingham, B15 2TT, UK; 7Moores Cancer Center and Department of Pathology, University of California San Diego, La Jolla, CA 92093, USA; 8Department of Pharmacology and Moores Cancer -
Guideline to Reference Gene Selection for Quantitative Real-Time PCR
BBRC Biochemical and Biophysical Research Communications 313 (2004) 856–862 www.elsevier.com/locate/ybbrc Guideline to reference gene selection for quantitative real-time PCR Aleksandar Radonicc,a Stefanie Thulke,a Ian M. Mackay,b Olfert Landt,d Wolfgang Siegert,a and Andreas Nitschea,c,d,* a Charite—Campus Charite Mitte, II. Medizinische Klinik mit Schwerpunkt Onkologie und Ha€matologie, Humboldt Universita€t, Berlin, Germany b Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Brisbane, Australia c Robert Koch Institut, Berlin, Germany d TIB MOLBIOL, Berlin, Germany Received 18 November 2003 Abstract Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and b-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that “Classical” reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant ex- pression in different tissues and following stimulation in CCRF-HSB-2 cells. -
Cell Type-Specific Analysis of Human Interactome and Transcriptome Shahin Mohammadi Purdue University
Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations January 2016 Cell Type-specific Analysis of Human Interactome and Transcriptome Shahin Mohammadi Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Recommended Citation Mohammadi, Shahin, "Cell Type-specific Analysis of Human Interactome and Transcriptome" (2016). Open Access Dissertations. 1371. https://docs.lib.purdue.edu/open_access_dissertations/1371 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated 12/26/2015 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Shahin Mohammadi Entitled CELL TYPE-SPECIFIC ANALYSIS OF HUMAN INTERACTOME AND TRANSCRIPTOME For the degree of Doctor of Philosophy Is approved by the final examining committee: Ananth Grama Wojciech Szpankowski Chair David Gleich Jennifer Neville Markus Lill To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Ananth Grama William Gorman, Assistant to the Department Head 11/1/2016 Approved by: Head of the Departmental Graduate Program Date CELL TYPE-SPECIFIC ANALYSIS OF HUMAN INTERACTOME AND TRANSCRIPTOME A Dissertation Submitted to the Faculty of Purdue University by Shahin Mohammadi In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016 Purdue University West Lafayette, Indiana ii I dedicate this thesis to my mom, whose role in my life I can not even begin to describe. -
Housekeeping Genes in Cancer
SHORT TECHNICAL REPORTS genes has deviated from the norm. Housekeeping genes in cancer: normalization Overexpression of ribosomal proteins of array data has been reported in certain cancers: colorectal (12), liver (13), and breast Anis H. Khimani1, Abner M. Mhashilkar2, Alvydas Mikulskis1, Michael (14). Recently, overexpression of O’Malley1, Jennifer Liao2, Eva E. Golenko1, Pat Mayer1, Sunil Chada2, ribosomal proteins L7a and L37 has Jeffrey B. Killian1, and Steven T. Lott1 been reported in prostate cancer tissues 1 2 when compared to a normal prostate PerkinElmer Life Sciences, Boston, MA and Introgen Therapeutics, Houston, TX, USA epithelial cell line (15). An examination of the expression of 15 different house- BioTechniques 38:739-745 (May 2005) keeping genes in colon cancer demon- strated substantial changes, particu- Biological maintenance of cells under variable conditions should affect gene expression larly in those coding for metabolic of only certain genes while leaving the rest unchanged. The latter, termed “housekeeping enzymes (16). Interestingly, this study genes,” by definition must reflect no change in their expression levels during cell develop- found little difference in ribosomal ment, treatment, or disease state anomalies. However, deviations from this rule have been proteins. In sharp contrast, exami- observed. Using DNA microarray technology, we report here variations in expression lev- nation of melanocytic lesions showed els of certain housekeeping genes in prostate cancer and a colorectal cancer gene therapy minimal variation between nevi and model system. To highlight, differential expression was observed for ribosomal protein genes melanoma (17). Housekeeping gene in the prostate cancer cells and β-actin in treated colorectal cells. -
Mammalian Genes Are Transcribed with Widely Different Bursting Kinetics
REPORTS transcribed from a gene and its protein product Mammalian Genes Are Transcribed are short-lived, the fluctuations of protein ex- pression should closely reflect the transcriptional with Widely Different Bursting Kinetics bursting kinetics (Fig. 1A). Although this ap- proach cannot capture abortive transcription events, it should reflect the production of mature David M. Suter,1* Nacho Molina,2* David Gatfield,1,3 Kim Schneider,1 mRNAs. We engineered a short-lived nuclear lu- Ueli Schibler,1†‡ Felix Naef2†‡ ciferase (NLS-luc) encoded by a short-lived mRNA, and fused it to blasticidin deaminase, separated In prokaryotes and eukaryotes, most genes appear to be transcribed during short periods by the 2A peptide of foot-and-mouth disease called transcriptional bursts, interspersed by silent intervals. We describe how such bursts virus, to generate two polypeptides from one cod- generate gene-specific temporal patterns of messenger RNA (mRNA) synthesis in mammalian ing sequence (18) (Fig. 1B). This cassette was cells. To monitor transcription at high temporal resolution, we established various gene trap cell integrated into three types of constructs: (i) a lines and transgenic cell lines expressing a short-lived luciferase protein from an unstable gene trap (GT) lentivector, allowing transcription mRNA, and recorded bioluminescence in real time in single cells. Mathematical modeling identified to be controlled by the endogenous locus at the gene-specific on- and off-switching rates in transcriptional activity and mean numbers of insertion site (19) (the resulting cell lines were mRNAs produced during the bursts. Transcriptional kinetics were markedly altered by cis-regulatory dubbed “GT:gene name”); (ii) vectors conferring DNA elements. -
Cardiac SARS‐Cov‐2 Infection Is Associated with Distinct Tran‐ Scriptomic Changes Within the Heart
Cardiac SARS‐CoV‐2 infection is associated with distinct tran‐ scriptomic changes within the heart Diana Lindner, PhD*1,2, Hanna Bräuninger, MS*1,2, Bastian Stoffers, MS1,2, Antonia Fitzek, MD3, Kira Meißner3, Ganna Aleshcheva, PhD4, Michaela Schweizer, PhD5, Jessica Weimann, MS1, Björn Rotter, PhD9, Svenja Warnke, BSc1, Carolin Edler, MD3, Fabian Braun, MD8, Kevin Roedl, MD10, Katharina Scher‐ schel, PhD1,12,13, Felicitas Escher, MD4,6,7, Stefan Kluge, MD10, Tobias B. Huber, MD8, Benjamin Ondruschka, MD3, Heinz‐Peter‐Schultheiss, MD4, Paulus Kirchhof, MD1,2,11, Stefan Blankenberg, MD1,2, Klaus Püschel, MD3, Dirk Westermann, MD1,2 1 Department of Cardiology, University Heart and Vascular Center Hamburg, Germany. 2 DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck. 3 Institute of Legal Medicine, University Medical Center Hamburg‐Eppendorf, Germany. 4 Institute for Cardiac Diagnostics and Therapy, Berlin, Germany. 5 Department of Electron Microscopy, Center for Molecular Neurobiology, University Medical Center Hamburg‐Eppendorf, Germany. 6 Department of Cardiology, Charité‐Universitaetsmedizin, Berlin, Germany. 7 DZHK (German Centre for Cardiovascular Research), partner site Berlin, Germany. 8 III. Department of Medicine, University Medical Center Hamburg‐Eppendorf, Germany. 9 GenXPro GmbH, Frankfurter Innovationszentrum, Biotechnologie (FIZ), Frankfurt am Main, Germany. 10 Department of Intensive Care Medicine, University Medical Center Hamburg‐Eppendorf, Germany. 11 Institute of Cardiovascular Sciences,