Flow Cytometric Analysis of RNA Synthesis by Detection of Bromouridine Incorporation

Jørgen K. Larsen1, Peter Østrup Jensen1, Jacob Larsen1

1 The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.12
DOI:  10.1002/0471142956.cy0712s12
Online Posting Date:  May, 2001
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Abstract

RNA synthesis has traditionally been investigated by a laborious and time‐consuming radiographic method involving incorporation of tritiated uridine. Now a faster non‐radioactive alternative has emerged, based on immunocytochemical detection. This method utilizes the brominated RNA precursor bromouridine, which is taken into a cell, phosphorylated, and incorporated into nascent RNA. The BrU‐substituted RNA is detected by permeabilizing the cells and staining with certain anti‐BrdU antibodies. This dynamic approach yields information complementing that provided by cellular RNA content analysis at a given time and may be of value in studies of cellular activation and gene expression.

     
 
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Table of Contents

  • Basic Protocol 1: Bivariate Analysis of DNA Content AND BrU Incorporation in Nuclei
  • Alternate Protocol 1: Bivariate Analysis of DNA Content and BrU Incorporation in Nuclei, Including Nucleoli
  • Alternate Protocol 2: Bivariate Analysis of DNA Content and BrU Incorporation in Cells
  • Alternate Protocol 3: Bivariate Analysis of Cell Surface Antigen Expression and BrU Incorporation
  • Support Protocol 1: Fluorescence Microscopy of Subcellular Distribution of BrU
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Bivariate Analysis of DNA Content AND BrU Incorporation in Nuclei

  Materials
  • Cells to be labeled and appropriate serum‐containing tissue culture medium ( appendix 3B)
  • 100 mM BrU stock solution (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A; store up to 2 months at 4°C)
  • Landberg's lysis buffer (see recipe)
  • 100% methanol, –20°C
  • PBS ( appendix 2A) containing 0.01% (v/v) Nonidet P‐40 (NP‐40)
  • Anti‐BrdU antibody (see recipe)
  • Polyclonal FITC‐conjugated rabbit anti‐mouse immunoglobulins (F[ab′] 2 fragment, Dako; protect from light during storage and handling)
  • PBS ( appendix 2A) containing 5% normal rabbit serum (Dako)
  • 50 µg/ml propidium iodide (PI) in PBS (store up to several months at 4°C; protect from light during storage and handling)
  • 20 mg/ml RNase (ribonuclease I‐A, Sigma) in PBS (store up to 2 weeks at 4°C; optional)
  • Green fluorescence standard microspheres (e.g., 1.7‐µm microspheres, Fluoresbrite 17687, Polysciences)
  • Unfixed chicken or trout erythrocytes stained with PI (see units 7.5 & 7.6)
  • Sample tubes as required for flow cytometer
  • Refrigerated centrifuge, 4°C
  • Flow cytometer with 488‐nm argon‐laser excitation and emission detectors for FITC fluorescence (525 ± 20 nm band‐pass) and PI fluorescence (630 ± 20 nm band‐pass)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Alternate Protocol 1: Bivariate Analysis of DNA Content and BrU Incorporation in Nuclei, Including Nucleoli

  • Otto's cell pretreatment buffer (see recipe)

Alternate Protocol 2: Bivariate Analysis of DNA Content and BrU Incorporation in Cells

  • 1% paraformaldehyde (see recipe), cold
  • 70% ethanol

Alternate Protocol 3: Bivariate Analysis of Cell Surface Antigen Expression and BrU Incorporation

  • R‐phycoerythrin‐conjugated monoclonal antibody, specific for cell surface antigen of choice (protect from light during storage and handling)
  • R‐phycoerythrin‐conjugated negative control antibody of same isotype
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS ( appendix 2A) containing 0.1% (w/v) bovine serum albumin (BSA)
  • PBS ( appendix 2A) containing 1% (w/v) paraformaldehyde and 0.05% (v/v) Nonidet P‐40 (NP‐40)
  • PBS ( appendix 2A) containing 1% (w/v) glycine
  • FITC‐conjugated anti‐BrU antibody (e.g., Becton Dickinson 7583)
  • PBS ( appendix 2A) containing 0.1% (w/v) BSA and 0.1% (v/v) NP‐40
  • Standard microspheres (FITC, R‐phycoerythrin, and blank) for compensation of spectral emission overlap (e.g., Calibrite Beads, Becton Dickinson)

Support Protocol 1: Fluorescence Microscopy of Subcellular Distribution of BrU

  • Embedding medium (e.g., Fluoromount‐G, Southern Biotechnology Associates) containing 0.5 µg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI; e.g., Serva)
  • Cytocentrifuge with containers of, e.g., 1 ml, 30 mm2 size (Zytokammer, Hettich‐Zentrifugen)
  • Fluorescence microscope with filter sets for FITC and DAPI fluorescence
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Figures

Videos

Literature Cited

Literature Cited
   Carayon, P. and Bord, A. 1992. Identification of DNA‐replicating lymphocyte subsets using a new method to label the bromodeoxyuridine incorporated into the DNA. J. Immunol. Methods 147:225‐230.
   Dolbeare, F. 1995. Bromodeoxyuridine: A diagnostic tool in biology and medicine. Part 1: Historical perspectives, histochemical methods and cell kinetics. Histochem. J. 27:339‐369.
   Haider, S.R., Juan, G., Traganos, F., and Darzynkiewicz, Z. 1997. Immunoseparation and immunodetection of nucleic acids labeled with halogenated nucleotides. Exp. Cell Res. 234:498‐506.
   Jackson, D.A., Iborra, F.J., Manders, E.M.M., and Cook, P.R. 1998. Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol. Biol. Cell 9:1523‐1536.
   Jensen, P.Ø., Larsen, J., Christiansen, J., and Larsen, J.K. 1993a. Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies. Cytometry 14:455‐458.
   Jensen, P.Ø., Larsen, J., and Larsen, J.K. 1993b. Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen. Acta Oncol. 32:521‐524.
   Landberg, G. and Roos, G. 1991. Antibodies to proliferating cell nuclear antigen (PCNA) as S‐phase specific probes in flow cytometric cell cycle analysis. Cancer Res. 51:4570‐4575.
   Li, X., Patel, R., Melamed, M.R., and Darzynkiewicz, Z. 1994. The cell cycle effects and induction of apoptosis by 5‐bromouridine in cultures of human leukemic MOLT‐4 and HL‐60 cell lines and mitogen stimulated normal lymphocytes. Cell Prolif. 27:307‐320.
   Murakami, T., Li, X., Gong, J., Bhatia, U., Traganos, F., and Darzynkiewicz, Z. 1995. Induction of apoptosis by 5‐azacytidine: Drug concentration‐dependent differences in cell cycle specificity. Cancer Res. 55:3093‐3098.
   Otto, F. 1990. DAPI staining of fixed cells for high‐resolution flow cytometry of nuclear DNA. Methods Cell Biol. 33:105‐110.
   Wansink, D.G., Nelissen, R.L.H., and de Jong, L. 1994. In vitro splicing of pre‐mRNA containing bromouridine. Mol. Biol. Rep. 19:109‐113.
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