title: Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice.
abstract: A modified polymerase chain reaction (PCR) assay for analysis of cytokine gene expression from reverse-transcribed (R/T) RNA obtained from small numbers of cells is described in detail. This method employs a previously described dot-blot format and utilizes a target specific radioactive oligonucleotide probe which hybridizes to the PCR amplified product, thus increasing both specificity and sensitivity. This obviates the need for repeated electrophoresis gels and easily accommodates large experiments (e.g., numerous samples or kinetic studies), using small amounts of RNA from low cell numbers. Manipulation of many samples is further enhanced with the use of a PCR thermocycler, which like the dot-blot apparatus is designed in a 96-well format. We describe the use of the house-keeping enzyme hypoxanthine phosphoribosyltransferase (HPRT) as an internal standard, which is especially suitable since its range of detectability of expression is similar to that of the cytokines under test. This enables one to obtain an accurate measure of losses or degradation of RNA, as well as controlling for efficiency of the R/T and PCR reactions. These reactions are further controlled by inclusion of a standard curve consisting of a titration of a known amount of RNA from a cell line expressing the cytokine under test. As well as controlling for the R/T-PCR, this standard curve also enables one to obtain a semi-quantitative measure of cytokine expression by different cell populations during an immune response. We show that this method can be used successfully for studying differential expression of IL-10 in different microenvironments during infection of mice with Schistosoma mansoni.
authors.affiliation: Department of Immunology, DNAX Research Institute for Molecular and Cellular Biology Inc., Palo Alto, CA 94304-1104.
authors.forename: E
authors.initials: E
authors.lastname: Murphy
authors.forename: S
authors.initials: S
authors.lastname: Hieny
authors.forename: A
authors.initials: A
authors.lastname: Sher
authors.forename: A
authors.initials: A
authors.lastname: O'Garra
mesh_terms: D000818:Animals; D001483:Base Sequence; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D007041:Hypoxanthine Phosphoribosyltransferase / Q000276:immunology; D016753:Interleukin-10 / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D012555:Schistosomiasis mansoni / Q000276:immunology*; D014158:Transcription, Genetic
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D016753:Interleukin-10; D012313:RNA; D007041:Hypoxanthine Phosphoribosyltransferase