Detection of cytokine mRNA in unfractionated peripheral blood by reverse transcriptase polymerase chain reaction.
OBJECTIVE: To detect cytokine gene expression in unfractionated peripheral blood by reverse transcriptase-polymerase chain reaction (RT-PCR). DESIGN: Prospective study. SETTING: University hospital, Japan. SUBJECTS: 3 healthy volunteers and 3 severely infected patients. INTERVENTIONS: 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 reverse transcripted, and interleukin-1 beta (IL-1beta) and tumour necrosis factor (TNF) cDNA were selectively amplified by synthetic primers with PCR. MAIN OUTCOME MEASURES: Establishment of cytokine gene expression in unfractionated peripheral blood. RESULTS: About 10 microg of total RNA was obtained from a 0.5 ml sample of blood. IL-1beta and TNF mRNA were not detected in blood from healthy volunteers, though they were detected in patients with severe infection. CONCLUSION: This method avoids artefactual gene activation and may be applicable to monitoring cytokine gene expression in various pathophysiological states.