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.
Faculty of Medicine, St. Joseph University, Beirut, Lebanon. nada.aladdin@usj.edu.lb
Nada
N
Alaaeddine
Claudette
C
De Montigny
Mbark
M
Sadouk
D005260:Female; D015870:Gene Expression; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000235:genetics*; D012016:Reference Values; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha