Detection of cytokine mRNA expression by PCR.
Assessment of cytokine expression during an immune response is a critical requirement for studies of basic immune mechanisms and the pathogenesis of disease. As an alternative to measuring cytokine protein levels, analysis of cytokine mRNA may be appropriate, because production of many cytokines is primarily transcriptionally regulated. Therefore, the level of cytokine mRNA present in a sample may serve as a good estimate of the level of cytokine protein present. This unit outlines the amplification of IL-2 mRNA from clinical biopsy specimens by the polymerase chain reaction (PCR), but the approach can be readily modified to study any cytokine RNA of interest in biopsies or small samples. In the Basic Protocol, cDNA is synthesized from the sample RNA by reverse transcriptase; to control for experimental variation in both the reverse transcription and PCR amplification steps, an internal control RNA is included in the reaction. Together, the reverse-transcribed cDNAs are amplified by PCR.
James
Stephen P
SP
University of Maryland, Baltimore, Maryland.
D000818:Animals; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry
D018076:DNA, Complementary; C508594:IL2 protein, human; D007376:Interleukin-2; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase