Analysis of differentially regulated mRNAs in monocytic cells induced by in vitro stimulation.
Macrophages are known to be effector cells in several granulomatous disorders. However, little is known about granuloma-associated up- or downregulation of genes in these cells. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) is an attractive method for the detection of differentially expressed genes. Although this method entails a number of drawbacks, its application to rare and limited amounts of clinical samples is still convenient. In this study, we introduce a screening procedure for detecting differentially regulated sequence tags in samples of patients suffering from granulomatous diseases. We applied DDRT-PCR in a multiple and complex comparison of expressed sequence tags in response to various granuloma-associated stimuli. The histiocytic cell line U937 was used as a model. The cells had been stimulated with granuloma-associated agents such as Mycobacterium tuberculosis, BeSO4, lipopolysaccharide, or HgS and unspecific stimuli such as phorbol myristate acetate, phytohemagglutinin, Zymosan, and Latex. Comparative analysis of 2237 sequence tags obtained from 55 primer combinations revealed 22.4% differentially amplified PCR products. Notably, only 8.0% of the differentially expressed sequence tags showed an association restricted to in vitro cultivation in the presence of M. tuberculosis, lipopolysaccharide, BeSO4, and/or HgS, while 1.0-1.9% of the tags were altered exclusively as a consequence of stimulation with one of the granuloma-associated agents. Our data provide evidence that this strategy may function as a preselection for appropriate primer combinations to discover sequence tags which could be specifically associated with granulomatous disorders. This approach could shorten laborious screening, save consumption of valuable and rare samples, and could reduce the number of false-positive results.
Gaede
K I
KI
Medical Hospital, Research Center Borstel, Germany. kgaede@fz-borstel.de
Mamat
U
U
Schlaak
M
M
Müller-Quernheim
J
J
D002460:Cell Line; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D006099:Granuloma / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D007378:Interleukins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma