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
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.