Detection and analysis of cytokine mRNA in tissues and cell lines.
Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the understanding of host responses to a variety of immunological insults. The detection of cytokines by bioassay or immunoassay provides data from in vitro experiments and can be used to determine the circulating plasma levels of particular cytokines. However, many investigations require the detection of cytokines in small numbers of cells or directly from tissue samples. It is in this particular area that detection and analysis of cytokine gene expression by use of the sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) has application. The purpose of these protocols is to provide a practical guide to the detection of cytokine gene expression using RT-PCR techniques. Procedures for analysing mRNA expression in cell culture, cell suspensions and tissue samples will be described and pitfalls and caveats concerning the techniques will be discussed. These protocols should provide a starting point for the development of RT-PCR technology in laboratories with only limited molecular experience.
Division of Immunobiology, The National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, UK. bwalker@nibsc.ac.uk
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D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D015870:Gene Expression; D007091:Image Processing, Computer-Assisted; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger; D004996:Ethidium