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Detection of cytokine mRNA in unfractionated peripheral blood by reverse transcriptase polymerase chain reaction
1Department of Surgery, Keio University School of Medicine, Shinnjuku-ku, Tokyo, Japan.
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.