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).


METHODS
Prospective study.


METHODS
University hospital, Japan.


METHODS
3 healthy volunteers and 3 severely infected patients.


METHODS
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.


METHODS
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.


CONCLUSIONS
This method avoids artefactual gene activation and may be applicable to monitoring cytokine gene expression in various pathophysiological states.
Shimizu
S
S
Department of Surgery, Keio University School of Medicine, Shinnjuku-ku, Tokyo, Japan.
Ueda
M
M
Ozawa
S
S
Endo
M
M
Wakabayashi
G
G
Kitajima
M
M
D000328:Adult; D000368:Aged; D001424:Bacterial Infections / Q000097:blood*; D001483:Base Sequence; D005260:Female; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000032:analysis / Q000235:genetics*; D007963:Leukocytes, Mononuclear; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012333:RNA, Messenger / Q000032:analysis* / Q000097:blood; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000235:genetics
D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha