Quantitative PCR reveals increased levels of tumor necrosis factor-alpha mRNA in peripheral blood mononuclear cells of multiple sclerosis patients during relapses.
Quantification of tumor necrosis factor-alpha (TNF-alpha) mRNA in peripheral blood mononuclear cells (PBMC) could provide information about disease activity in multiple sclerosis (MS); however, specific competitive methods must be utilized. A competitor cDNA, having the same sequence of the target TNF-alpha cDNA, a part from an internal 49-bp deletion, was generated and used to set-up a quantitative polymerase chain reaction (PCR) to quantify mRNA of TNF-alpha. Competitor and target were co-amplified using the same primers. The rates of generation of competitor and target TNF-alpha conformed closely to the prediction of the mathematical model, and a high level of accuracy and reproducibility was achieved. The method was applied to quantify TNF-alpha mRNA in PBMC of normal subjects and multiple sclerosis (MS) patients both during clinical relapses and remissions. A statistically significant higher level of TNF-alpha mRNA was detected during relapses than during remissions. High levels of TNF-alpha mRNA were found in 44% of relapses and 12% of samples during remissions, suggesting that TNF-alpha mRNA synthesis is abnormal in MS.
Bertolotto
A
A
Centro Sclerosi Multipla, Divisione Universitaria di Neurologia, H.S. Luigi, Orbassano, Italy. nsglb@tin.it
Malucchi
S
S
Capobianco
M
M
Manzardo
E
E
Guastamacchia
G
G
Milano
E
E
Audano
L
L
Mutani
R
R
D000328:Adult; D001667:Binding, Competitive; D016022:Case-Control Studies; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D016015:Logistic Models; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis*; D012008:Recurrence; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha