Real-time reverse transcription-polymerase chain reaction quantification of tumor necrosis factor alpha messenger in human leukocytes.

BACKGROUND: Tumor necrosis factor (TNF)-alpha plays a significant role in the pathogenesis of several inflammatory conditions. Several studies confirmed high TNF-alpha plasma protein levels in such conditions and highlighted the utility of TNF-alpha as a biomarker for disease progression or response to biological therapy. We aimed to provide a novel molecular technique for the measurement of TNF-alpha.
METHODS: Quantitative assay for the measurement of TNF-alpha messenger ribonucleic acid (mRNA) was done using real time reverse transcription polymerase chain reaction (RT-PCR).
RESULTS: The test was specific, sensitive, and efficient and possessed a wide scale of linearity. Intrassay and interassay CVs were equivalent to those observed in enzyme-linked immunosorbent assay methods for plasmatic TNF-alpha. Data from 11 healthy adults recruited in an attempt to establish reference values showed an average of 109.4 ng of U937 RNA (range: 67.0 - 173.5). Circulating TNF-alpha protein was not detected by ELISA in any of the 11 subjects.
CONCLUSIONS: The test may be useful for clinical studies and in research investigating TNF-alpha gene expression both in physiologic and pathologic situations, although these pilot findings need to be confirmed through larger studies.