DETECTION OF CYTOKINE MESSENGER-RNA IN UNFRACTIONATED PERIPHERAL-BLOOD BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION

Citation
S. Shimizu et al., DETECTION OF CYTOKINE MESSENGER-RNA IN UNFRACTIONATED PERIPHERAL-BLOOD BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION, The European journal of surgery, 163(7), 1997, pp. 487-492
Citations number
27
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Surgery
ISSN journal
1102-4151
Volume
163
Issue
7
Year of publication
1997
Pages
487 - 492
Database
ISI
SICI code
1102-4151(1997)163:7<487:DOCMIU>2.0.ZU;2-Z
Abstract
Objective: To detect cytokine gene expression in unfractionated periph eral blood by reverse transcriptase-polymerase chain reaction (RT-PCR) . Design: Prospective study. Setting: University hospital, Japan. Subj ects: 3 healthy volunteers and 3 severely infected patients. Intervent ions: Peripheral blood was obtained and total RNA was extracted from 0 .5 ml unfractionated whole blood with a 4 M guanidinium isothiocyanate mixture, 0.2 M sodium acetate, phenol, and chloroform. The mRNA was r everse transcripted, and interleukin-1 beta (IL-1 beta) and tumour nec rosis factor (TNF) cDNA were selectively amplified by synthetic primer s with PCR. Main outcome measures: Establishment of cytokine gene expr ession in unfractionated peripheral blood. Results: About 10 mu g of t otal RNA was obtained from a 0.5 ml sample of blood. IL-1 beta and TNF mRNA were not detected in blood from healthy volunteers, though they were detected in patients with severe infection. Conclusion: This meth od avoids artefactual gene activation and may be applicable to monitor ing cytokine gene expression in various pathophysiological states.