Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method

G. A. Bishop, K. L. Rokahr, M. Lowes, P. H. Mcguinness, J. Napoli, D. J. DeCruz, W. Y. Wong, G. W. Mccaughan

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small samples. The aim of this work was to investigate the utility of a non-competitive RT-PCR which used external standards to quantitate TNF-α mRNA in liver biopsy specimens from liver transplant patients. It involved removal of aliquots from the PCR reaction at successive cycles, followed by dot-blotting of the samples onto nylon membrane and hybridization with a radioactively-labelled internal probe. Phosphorimage analysis of the labelled membranes allowed quantitation of the relative amount of PCR product at successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplification. The slopes of these lines showed the efficiency of amplification, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-α in two separate PCR amplifications showed low inter-assay variability (r2 = 0.98). Comparison of two separate cDNA syntheses also showed good correlation (r2 = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that variation in efficiency of cDNA synthesis is likely to contribute as much or more to variability of the analysis as variations in PCR amplification.

Original languageEnglish (US)
Pages (from-to)142-147
Number of pages6
JournalImmunology and Cell Biology
Volume75
Issue number2
StatePublished - 1997
Externally publishedYes

Fingerprint

Biopsy
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Liver
Amplification
Cytokines
Polymerase Chain Reaction
Messenger RNA
Complementary DNA
Membranes
Transplants
Nylons
Assays

Keywords

  • cytokines
  • PCR
  • quantitation
  • transplantation

ASJC Scopus subject areas

  • Immunology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Bishop, G. A., Rokahr, K. L., Lowes, M., Mcguinness, P. H., Napoli, J., DeCruz, D. J., ... Mccaughan, G. W. (1997). Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method. Immunology and Cell Biology, 75(2), 142-147.

Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method. / Bishop, G. A.; Rokahr, K. L.; Lowes, M.; Mcguinness, P. H.; Napoli, J.; DeCruz, D. J.; Wong, W. Y.; Mccaughan, G. W.

In: Immunology and Cell Biology, Vol. 75, No. 2, 1997, p. 142-147.

Research output: Contribution to journalArticle

Bishop, GA, Rokahr, KL, Lowes, M, Mcguinness, PH, Napoli, J, DeCruz, DJ, Wong, WY & Mccaughan, GW 1997, 'Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method', Immunology and Cell Biology, vol. 75, no. 2, pp. 142-147.
Bishop, G. A. ; Rokahr, K. L. ; Lowes, M. ; Mcguinness, P. H. ; Napoli, J. ; DeCruz, D. J. ; Wong, W. Y. ; Mccaughan, G. W. / Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method. In: Immunology and Cell Biology. 1997 ; Vol. 75, No. 2. pp. 142-147.
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