title: Detection of cytokine mRNA in unfractionated peripheral blood by reverse transcriptase polymerase chain reaction.
abstract: 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.
authors.affiliation: Department of Surgery, Keio University School of Medicine, Shinnjuku-ku, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
authors.forename: M
authors.initials: M
authors.lastname: Ueda
authors.forename: S
authors.initials: S
authors.lastname: Ozawa
authors.forename: M
authors.initials: M
authors.lastname: Endo
authors.forename: G
authors.initials: G
authors.lastname: Wakabayashi
authors.forename: M
authors.initials: M
authors.lastname: Kitajima
mesh_terms: 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
chemical_list: D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha