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Titolo:
QUANTITATION OF CHANGES IN P-0 MESSENGER-RNA BY POLYMERASE-CHAIN-REACTION IN PRIMARY CULTURED SCHWANN-CELLS STIMULATED BY AXOLEMMA-ENRICHEDFRACTION
Autore:
CLIVE DR; LOPEZ TJ; DEVRIES GH;
Indirizzi:
EDWARD HINES JR DEPT VET AFFAIRS HOSP,RES SERV HINES IL 60141 EDWARD HINES JR DEPT VET AFFAIRS HOSP,RES SERV HINES IL 60141 LOYOLA UNIV,MED CTR,DEPT CELL BIOL NEUROBIOL & ANAT MAYWOOD IL 60153
Titolo Testata:
Journal of neuroscience methods
fascicolo: 1-2, volume: 81, anno: 1998,
pagine: 25 - 34
SICI:
0165-0270(1998)81:1-2<25:QOCIPM>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
MESSENGER-RNA; REVERSE TRANSCRIPTION; PERIPHERAL MYELIN; EXPRESSION; PROTEIN; GENE; QUANTIFICATION; PROLIFERATION; ABSORBENCY; RATIOS;
Keywords:
DIFFERENTIATION; SCHWANN CELLS; MYELIN GENE EXPRESSION; QUANTITATIVE RT-PCR; AXOLEMMA-ENRICHED FRACTION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
37
Recensione:
Indirizzi per estratti:
Citazione:
D.R. Clive et al., "QUANTITATION OF CHANGES IN P-0 MESSENGER-RNA BY POLYMERASE-CHAIN-REACTION IN PRIMARY CULTURED SCHWANN-CELLS STIMULATED BY AXOLEMMA-ENRICHEDFRACTION", Journal of neuroscience methods, 81(1-2), 1998, pp. 25-34

Abstract

A requirement for large numbers of primary culture cells has frequently restricted investigations of gene expression in glial cells. We have developed a non-radio active method based on reverse transcription-polymerase chain reaction (RT-PCR) to accurately assess small changes in the expression of the myelin specific gene P-0 in Schwann cells. Using axolemma-enriched fraction (AEF) as an inducing agent, we demonstrate that RT-PCR can be used to detect 4-8-fold increases in P-0 mRNA levels occurring in a time and dose dependent manner, utilizing only 250000 cells per assay. Initial experiments used an in vitro transcribed RNA for P-0 constructed with a 300 bp deletion for quantitation by competitive RT-PCR. Relative quantitation by co-amplification of the housekeeping gene glyceraldehyde-phosphate dehydrogenase nas established and provided similar results. Product evaluation was enhanced 50-100-fold by the incorporation of primers labelled with biotin at the 5' end,allowing for the sensitive detection of PCR product by enhanced chemiluminescence and autoradiography. This technique provides sensitivity to detect and evaluate picogram amounts of DNA. Our results validate the assay for P-0 gene expression and indicate that the technique should facilitate the study of multiple genes of interest in glial cell systems. (C) 1998 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/07/20 alle ore 07:20:14