Abstract
We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR product visualization of a multiwelled agarose gel, stained with SYBR Green I dye. The transcriptional assay was unaffected by solvents (dimethyl sulfoxide and methanol) routinely used in high-throughput drug screens at concentrations required for compound solubilization. Furthermore, it has been used successfully for the investigation of differential mRNA expression levels of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells (a human monocytic cell line) and the identification of specific IL-1 beta transcriptional inhibitors.
MeSH terms
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Benzothiazoles
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Cell Line
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DNA Primers
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Diamines
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Electrophoresis, Agar Gel
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Fluorescent Dyes
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Gene Expression Regulation
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Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
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Humans
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Imidazoles / pharmacology
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Interleukin-1 / genetics
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Kinetics
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Lactones / pharmacology
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Lipopolysaccharides / pharmacology
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Macrolides
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Monocytes / metabolism
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Organic Chemicals*
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Polymerase Chain Reaction / methods*
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Quinolines
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RNA, Messenger / analysis*
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RNA, Messenger / genetics
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RNA, Messenger / isolation & purification
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Solvents
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Thiazoles / pharmacology
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Transcription, Genetic*
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Tumor Necrosis Factor-alpha / genetics
Substances
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Benzothiazoles
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DNA Primers
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Diamines
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Fluorescent Dyes
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Imidazoles
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Interleukin-1
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Lactones
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Lipopolysaccharides
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Macrolides
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Organic Chemicals
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Quinolines
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RNA, Messenger
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Solvents
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Thiazoles
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Tumor Necrosis Factor-alpha
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SYBR Green I
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6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo(2,1-b)thiazole
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Glyceraldehyde-3-Phosphate Dehydrogenases
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monorden