Quantitative analysis of immune-mediated stimulation of tumor necrosis factor-alpha in macrophages measured at the level of mRNA and protein synthesis.
Expression of cytokines such as tumor necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS) has been associated with inflammatory and regulatory immune reactions. Antigen-presenting cells, especially macrophages, play a central role in directing immune responses by synthesizing different cytokines. For the analysis of cytokine synthesis, we compared quantitative changes in mRNA and protein synthesis of TNF-alpha in RAW 264.7 cells stimulated with 0.1 ng/ml LPS. TNF-alpha mRNA was quantified using the LightCycler SYBR Green technology (Idaho Technology, Inc., Salt Lake City, Utah, USA). RAW 264.7 cells showed a basal TNF-alpha mRNA expression which increased approximately sixfold after 2 h of stimulation with LPS. TNF-alpha synthesis was analyzed at the protein level using a mouse-specific sandwich enzyme-linked immunosorbent assay (ELISA) and indicated a 56-fold increase in TNF-alpha protein concentration after 4 h. Thus, real-time polymerase chain reaction (PCR) is a sensitive and rapid method for quantitative determination of LPS-induced TNF-alpha expression. However, it requires extremely robust reaction parameters to be reliable for accurate quantification.
Weghofer
M
M
Institute for Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.
Karlic
H
H
Haslberger
A
A
D000199:Actins / Q000235:genetics; D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics*
D000199:Actins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha