Detection of Cytokine mRNA Expression by PCR

Stephen P. James1

1 University of Maryland, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.23
DOI:  10.1002/0471142735.im1023s10
Online Posting Date:  May, 2001
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Abstract

Assessment of cytokine expression during an immune response is a critical requirement for studies of basic immune mechanisms and the pathogenesis of disease. As an alternative to measuring cytokine protein levels, analysis of cytokine mRNA may be appropriate, because production of many cytokines is primarily transcriptionally regulated. Therefore, the level of cytokine mRNA present in a sample may serve as a good estimate of the level of cytokine protein present. This unit outlines the amplification of IL‐2 mRNA from clinical biopsy specimens by the polymerase chain reaction (PCR), but the approach can be readily modified to study any cytokine RNA of interest in biopsies or small samples. In the , cDNA is synthesized from the sample RNA by reverse transcriptase; to control for experimental variation in both the reverse transcription and PCR amplification steps, an internal control RNA is included in the reaction. Together, the reverse‐transcribed cDNAs are amplified by PCR.

     
 
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Table of Contents

  • Basic Protocol 1: Quantitation of IL‐2 mRNA Expression
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitation of IL‐2 mRNA Expression

  Materials
  • Internal control: transcription plasmid vector containing cDNA of interest
  • Clinical biopsy samples
  • Guanidinium isothiocyanate buffer (unit 10.11)
  • 50 U/µl RNasin (Promega)
  • recipe5 × Reverse transcriptase buffer
  • 0.5 mg/ml oligo(dT) 16 (Sigma)
  • 1 mg/ml bovine serum albumin, acetylated (BSA, Sigma #B‐8894)
  • 4dNTP mix (unit 10.20)
  • 200 U/µl Mo‐MuLV reverse transcriptase (GIBCO/BRL)
  • 10× amplification buffer (unit 10.20)
  • Amplification primers: 20 µM each 5′ and 3′ primers specific for cytokine of interest (Tables 10.23.1 & 10.23.2)
  • 5 U/µl Taq DNA polymerase, diluted as in unit 10.20
  • Mineral oil
  • 3% agarose gel containing 1 µg/ml ethidium bromide (unit 10.4)
  • DNA molecular weight markers (unit 10.4)
  • 13 × 100–mm tissue grinder (Pyrex, Corning)
  • 1.5‐ml and 0.5‐ml microcentrifuge tubes, autoclaved
  • 65° and 39°C water baths
Additional reagents and equipment for preparation of RNA (unit 10.11), PCR (unit 10.20), and agarose gel electrophoresis (unit 10.4)CAUTION: When working with human blood, cells, or infectious agents, biosafety practices must be followed (see Chapter 7).NOTE: Use standard precautions when handling RNA samples. All buffers and sample dilutions are made in sterile water (see units 10.11 & 10.20 for guidelines).
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Figures

Videos

Literature Cited

Literature Cited
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   Mullin, G.E., Lazenby, A.J., Harris, M.L., Bayless, T.M., and James, S.P. 1992. Increased interleukin‐2 mRNA in the intestinal mucosal lesions of Crohn's disease but not ulcerative colitis. Gastroenterology 102:1620‐1627.
   Rappolee, D.A., Wang, A., Mark, D., and Werb, Z. 1989. Novel method for studying mRNA phenotypes in single or small numbers of cells. J. Cell. Biochem. 39:1‐11.
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Key Reference
   Mullin, et al., 1992. See above.
  Describes in detail the application of this method to quantitation of IL‐2 mRNA in intestinal biopsies, demonstrating the changes in IL‐2 levels during inflammatory disease.
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