A method for rapid analysis of peripheral blood mononuclear leukocyte cytokine mRNA

S. W. Bickler, Michael Heinrich, Michael Davey, M. W. Harrison, G. C. Bagby

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Interleukins, hematopoietic growth factors, and adhesion molecules mediate multiple components of the inflammatory response. Conventional methods for purification of cytokine-producing cells are time consuming and can, therefore, depending on the conditions of cell purification and the nature of the mRNA being purified, result in both over- and underestimates of mRNA content. Therefore, it has been difficult to quantify such factors in ways that are unambiguously reflective of in vivo cytokine production. We describe herein a method for such rapid preparation of peripheral blood mononuclear leukocytes (PBML) that amounts of cytokine mRNA derived from these cells, apart from readily quantifiable losses during RNA preparation, will be reflective of quantities in vivo. Small quantities (105) of normal PBML were isolated by density centrifugation of heparinized blood for 1.25 min in a capillary tube. Poly(A)+ RNA isolated by oligo(dT)-cellulose column chromatography was sufficient to reverse transcribe both antigen-specific T-cell receptor β-chain mRNA and interleukin 6(IL-6) mRNA and subsequently amplify the cDNA with Taq polymerase in reverse transcription-polymerase chain reactions (RT-PCR). Although IL-6 mRNA was not present in normal PBML, PBML incubated in culture medium for only 3 h contained approximately one molecule per cell. Because of its rapidity this technique will permit quantification of cytokine mRNA in the steady state and in clinical settings of inflammation. Because the method requires only a small quantity of blood it can be applied to clinical research studies involving children.

Original languageEnglish (US)
Pages (from-to)980-985
Number of pages6
JournalExperimental Hematology
Volume20
Issue number8
StatePublished - 1992

Fingerprint

Mononuclear Leukocytes
Cytokines
Messenger RNA
Interleukin-6
Taq Polymerase
Interleukins
T-Cell Antigen Receptor
Centrifugation
Reverse Transcription
Culture Media
Chromatography
Intercellular Signaling Peptides and Proteins
Complementary DNA
RNA
Inflammation
Polymerase Chain Reaction

Keywords

  • β-chain
  • cytokine
  • interleukin 6
  • mRNA
  • polymerase chain reaction
  • T-cell receptor

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

A method for rapid analysis of peripheral blood mononuclear leukocyte cytokine mRNA. / Bickler, S. W.; Heinrich, Michael; Davey, Michael; Harrison, M. W.; Bagby, G. C.

In: Experimental Hematology, Vol. 20, No. 8, 1992, p. 980-985.

Research output: Contribution to journalArticle

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