title: A method for obtaining simian immunodeficiency virus RNA sequences from laser capture microdissected and immune captured CD68+ and CD163+ macrophages from frozen tissue sections of bone marrow and brain.
abstract: Laser capture microdissection (LCM) is used to extract cells or tissue regions for analysis of RNA, DNA or protein. Several methods of LCM are established for different applications, but a protocol for consistently obtaining lentiviral RNA from LCM captured immune cell populations is not described. Obtaining optimal viral RNA for analysis of viral genes from immune-captured cells using immunohistochemistry (IHC) and LCM is challenging. IHC protocols have long antibody incubation times that increase risk of RNA degradation. But, immune capture of specific cell populations like macrophages without staining for virus cannot result in obtaining only a fraction of cells which are productively lentivirally infected. In this study we sought to obtain simian immunodeficiency virus (SIV) RNA from SIV gp120+ and CD68+ monocyte/macrophages in bone marrow (BM) and CD163+ perivascular macrophages in brain of SIV-infected rhesus macaques. Here, we report an IHC protocol with RNase inhibitors that consistently results in optimal quantity and yield of lentiviral RNA from LCM-captured immune cells.
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, MA, USA.
authors.forename: Jaclyn
authors.initials: J
authors.lastname: Mallard
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, MA, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Papazian
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, MA, USA; Department of Research and Development, Innate Pharma, Marseille, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Soulas
authors.affiliation: Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA; Department of Pathology Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Nolan
authors.affiliation: Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA; Department of Pathology Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Salemi
authors.affiliation: Department of Biology, Boston College, Chestnut Hill, MA, USA. Electronic address: kenneth.williams.3@bc.edu.
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Williams
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis; D001853:Bone Marrow / Q000276:immunology* / Q000821:virology; D001921:Brain / Q000276:immunology* / Q000821:virology; D007150:Immunohistochemistry; D061346:Laser Capture Microdissection; D008253:Macaca mulatta; D008264:Macrophages / Q000276:immunology* / Q000821:virology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D016097:Simian Acquired Immunodeficiency Syndrome / Q000235:genetics* / Q000276:immunology / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology; D000098902:CD163 Antigen; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D011956:Receptors, Cell Surface; D014759:Viral Envelope Proteins; D000098902:CD163 Antigen; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C069623:gp120 protein, Simian immunodeficiency virus
keywords: Immunohistochemistry (IHC); Laser capture microdissection (LCM); RNA; Simian immunodeficiency virus (SIV)