Quantitation of Human Heme Oxygenase (HO‐1) Copies by Competitive RT‐PCR

N.G. Abraham1, S.K. Shenouda1, A. Goodman1

1 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.8
DOI:  10.1002/0471140856.tx0908s12
Online Posting Date:  August, 2002
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Abstract

The authors have developed a system for quantifying human HO‐1 mRNA in samples limited in cell number and/or mRNA copies. Total RNA from human liver was used to develop the system. The RNA is reverse transcribed and amplified by PCR in a tube also containing an internal standard obtained by deleting 50 base pairs from the original human gene. After amplification the two templates are resolved and quantified. When the internal standard is present in the reaction mixture, the ratio of amplified sample to internal standard is proportional to the amount of sample RNA making it possible to calculate the number of specific mRNA molecules.

     
 
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Table of Contents

  • Basic Protocol 1: Quantitative RT‐PCR to Measure HO‐1 mRNA
  • Support Protocol 1: Distinguishing Between the Amplified Templates
  • Support Protocol 2: Competitive Versus Noncompetitive Amplification
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative RT‐PCR to Measure HO‐1 mRNA

  Materials
  • pCMV‐HHO‐1 plasmid (Yoshida et al., )
  • 1 U/µl EcoR47III and buffer (Promega)
  • 1 U/µl Bal31 nuclease and buffer
  • 2% (w/v) agarose gel ( appendix 3A)
  • 100‐bp DNA molecular weight marker VI (Boehringer Mannheim Biochemicals)
  • 10 mg/ml ethidium bromide
  • 0.5 M EDTA ( appendix 2A)
  • 1 U/ml T4 DNA ligase and buffer
  • JM109 E. coli transformation‐competent bacteria ( appendix 3A)
  • LB broth and plates or equivalent containing 10 mg/ml ampicillin ( appendix 3A)
  • 1:3 phenol/chloroform ( appendix 3A)
  • Qiagen miniprep kit
  • 1 U/ml HindIII restriction enzyme and buffer
  • 1% (w/v) low‐melting‐temperature agarose gel ( appendix 3A)
  • Tissue sample (e.g., human liver)
  • First‐Strand cDNA Synthesis Kit (Clontech Laboratories):
    • DEPC‐treated H 2O (unit 2.9)
    • 10 µM oligo(dT) 18
    • 0.1 M Tris⋅Cl, pH 8.3 ( appendix 2A)
    • 0.1 M KCl
    • 0.1 M MgCl 2
    • 100 mM dNTPs in sterile deionized H 2O (Promega)
    • 2 U/ml RNase inhibitor
    • 100 U/ml Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories)
  • 0.001% (w/v) gelatin
  • 1 µM sense primer: 5′‐CAG GCA GAG AAT GCT GAG TTC‐′ (Kutty et al. ; CER)
  • 1 µM antisense primer: 5′‐GAT GTT GAG CAG GAA CGC AGT‐3′ (Kutty et al., ; CER)
  • 2.5 U/µl Taq DNA polymerase (Stratagene)
  • 5 Ci/µl (185 GBq/µl) [α‐32P] dCTP (3000 Ci/mmol)
  • Mineral oil (optional)
  • 6% (w/v) 37.5:1 acrylamide/bisacrylamide gel ( appendix 3A)
  • 25 mM Tris⋅Cl, pH 8.3 ( appendix 2A)/200 mM glycine/1 mM EDTA
  • Thermocycler
  • Additional reagents and equipment for restriction endonuclease digestion, agarose gel electrophoresis, ethidium bromide staining, bacterial transformation, cell culture, phenol/chloroform extraction, determination of RNA purity by UV spectroscopy, polyacrylamide gel electrophoresis, plasmid amplification and purification ( appendix 3A), RNA isolation (Chomczynski and Sacchi, ; appendix 3A), and autoradiography ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Abraham, N.G. 1998. Quantitation of HO‐1 copies in human tissues by competitive RT/PCR. In Free Radicals and Antioxidant Protocols (D. Armstrong, ed.) Humana Press, Totowa, N.J.
   Abraham, N.G., Lavrovsky, Y., Schwartzman, M.L., Stoltz, R.A., Levere, R.D., Gerritsen, E., Shibahara, S., and Kappas, A. 1995. Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: Protective effect against heme and hemoglobin toxicity. Proc. Natl. Acad. Sci. U.S.A. 92:6798‐6802.
   Becker‐Andre, M. and Hahlbrock, K. 1989. Absolute mRNA quantitation using the polymerase chain reaction (PCR): A novel approach by a PCR aided transcript titration assay (PATTY). Nucl. Acids Res. 17:9437‐9441.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidium. Anal. Biochem. 162:156‐159.
   Cross, N.C. 1995. Quantitative PCR techniques and applications. Br. J. Haematol. 89:693‐697.
   Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H.F. 1990. Analysis of cytokines mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 87:2725‐2729.
   Goodman, A.I., Choudhury, M., da Silva, J‐L., Jiang, S., and Abraham, N.G. 1996. Quantitative measurement of heme oxygenase‐1 in the human renal adenocarcinoma. J. Cell. Biochem. 63:342‐348.
   Kutty, G., Hayden, B., Osawa, Y., Wiggert, B., Chader, G.J., and Kutty, R.K. 1992. Heme oxygenase: Expression in human retina and modulation by stress agents in a human retinoblastoma cell model system. Curr. Eye Res. 11:153‐160.
   Laniado‐Schwartzman, M., Abraham, N.G., Conners, M., Dunn, M.W., Levere, R.D., and Kappas, A. 1997. Heme oxygenase induction with attenuation of experimentally‐induced corneal inflammation. Biochem. Pharmacol. 53:1069‐1075.
   Levere, R.D., Martasek, P., Escalante, B., Schwartzman, M.L., and Abraham, N.G. 1990. Effect of Heme Arginate administration on BP in spontaneously hypertensive rats. J. Clin. Invest. 86:213‐219.
   Maines, M.D. 1997. The heme oxygenase system: A regulator of second messenger gases. Annu. Rev. Pharmacol. Toxicol. 37:517‐554.
   McCoubrey, W.K., Jr., Ewing, J.F., and Maines, M.D. 1992. Human heme oxygenase‐2: Characterization and expression of a full‐length cDNA and evidence suggesting that the two HO‐2 transcripts may differ by choice of a polyadenylation signal. Arch. Biochem. Biophys. 295:13‐20.
   Murphy, L.D., Herzog, C.E., Rudick, J.B., Fojo, A.T., and Bates, S.E. 1990. Use of polymerase chain reaction in the quantitation of mdr‐1 gene expression. Biochemistry 29:10351‐10356.
   Nath, K.A., Balla, G., Vercellotti, G.M., Balla, J., Jacob, H.S., Levitt, M.D., and Rosenberg, M.E. 1992. Induction of heme oxygenase is a rapid protective response in rhabdomyolysis in the rat. J. Clin. Invest. 90:267‐270.
   Otterbein, L.E., Bach, F.H., Alam, J., Soares, M., Tao, L.H., Wysk, M., Davis, R.J., Flavell, R.A., and Choi, A.M. 2000. Carbon monoxide has anti‐inflammmarory effects involving the mitogen‐activated protein kinase pathway. Nat. Med. 4:422‐428.
   Poss, K.D. and Tonegawa, S. 1997. Heme oxygenase‐1 is required for mammalian iron utilization. Proc. Natl. Acad. Sci. U.S.A. 94:10919‐10924.
   Quan, S., Yang, L., Abraham, N.G., and Kappas, A. 2001. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs. Proc. Natl. Acad. Sci. U.S.A. 98:12203‐8.
   Schacter, B.A. and Kurz, P. 1986. Alterations in microsomal drug metabolism and heme oxygenase activity in isolated hepatic parenchymal and sinusoidal cells in Murphy‐Sturn lymphosarcoma‐bearing rats. Clin. Invest. Med. 9:150‐155.
   Solangi, K., Sacerdoti, D., Goodman, A.I., Schwartzman, M.L., Abraham, N.G., and Lever, R.T. 1988. Differential effects of partial hepatectomy on hepatic and renal heme and cytochrome P450 metabolism. Am. J. Med. Sci. 296:387‐391.
   Wagener, F.A., da Silva, J.L., Farley, T., de Witte, T., Kappas, A., and Abraham, N.G. 1999. Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression. J. Pharmacol. Exp. Ther. 291:416‐23.
   Wang, A.M., Doyle, M.V., and Mark, D.F. 1989. Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 86:9717‐9721.
   Yachie, A., Niida, Y., Wada, T., Igarashi, N., Kaneda, H., Toma, T., Ohta, K., Kasahara, Y., and Koizumi, S. 1999. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase‐1 deficiency. J. Clin. Invest. 103:129‐135.
   Yang, L., Quan, S., and Abraham, N.G. 1999. Retrovirus‐mediated HO gene transfer into endothelial cells protect against oxidant‐induced injury. Am. J. Physiol.(part 1) 277:L127‐L133.
   Yoshida, T., Biro, P., Cohen, T., Muller, R.M., and Shibahara, S. 1988. Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur. J. Biochem. 171:457‐461.
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