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Titolo:
DETECTION OF IN-VIVO EXPRESSION OF INTERLEUKIN-10 USING A SEMIQUANTITATIVE POLYMERASE CHAIN-REACTION METHOD IN SCHISTOSOMA-MANSONI-INFECTEDMICE
Autore:
MURPHY E; HIENY S; SHER A; OGARRA A;
Indirizzi:
DNAX RES INST MOLEC & CELLULAR BIOL INC,DEPT IMMUNOL,901 CALIF AVE PALO ALTO CA 94304 DNAX RES INST MOLEC & CELLULAR BIOL INC,DEPT IMMUNOL,901 CALIF AVE PALO ALTO CA 94304 NIAID,PARASIT DIS LAB,IMMUNOL & CELL BIOL SECT BETHESDA MD 20892
Titolo Testata:
Journal of immunological methods
fascicolo: 2, volume: 162, anno: 1993,
pagine: 211 - 223
SICI:
0022-1759(1993)162:2<211:DOIEOI>2.0.ZU;2-7
Fonte:
ISI
Lingua:
ENG
Soggetto:
MESSENGER-RNA; CYTOKINE PRODUCTION; CELLS;
Keywords:
SCHISTOSOMA-MANSONI; T-HELPER; INTERLEUKIN; REVERSE TRANSCRIPTION; POLYMERASE CHAIN REACTION, SEMIQUANTITATIVE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
18
Recensione:
Indirizzi per estratti:
Citazione:
E. Murphy et al., "DETECTION OF IN-VIVO EXPRESSION OF INTERLEUKIN-10 USING A SEMIQUANTITATIVE POLYMERASE CHAIN-REACTION METHOD IN SCHISTOSOMA-MANSONI-INFECTEDMICE", Journal of immunological methods, 162(2), 1993, pp. 211-223

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 samplesis 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 amountof 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 toobtain a semi-quantitative measure of cytokine expression by different cell populations during an immune response. We show that this methodcan be used successfuly for studying differential expression of IL-10in different microenvironments during infection of mice with Schistosoma mansoni.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/11/20 alle ore 04:07:28