title: Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals.
abstract: BACKGROUND
  As RNA is labile, we investigated whether circulating RNA in human plasma may be present in a particle-associated form.
  
  
  METHODS
  Blood was collected from 27 healthy individuals and 16 hepatocellular carcinoma (HCC) patients. The plasma from each individual was processed by two means: filtration through filters with different pore sizes (from 5 microm to 0.22 microm) and ultracentrifugation. We assessed plasma RNA content by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and plasma DNA by a real-time quantitative PCR assay for the beta-globin gene.
  
  
  RESULTS
  The plasma GAPDH mRNA concentrations in the healthy individuals were significantly different in every pair of these filter sizes (P <0.05 for each pair). Overall, the plasma GAPDH mRNA concentration was higher by a median of 15-fold (interquartile range, 10- to 24-fold) in the paired unfiltered sample than in the sample filtered through a 0.22 microm filter. In contrast, no significant difference was seen in beta-globin DNA concentrations among different pore-size-filtered plasma samples (P = 0.455). Similarly, a significant difference was observed for RNA, but not DNA, between unfiltered plasma and ultracentrifuged plasma (P <0.05). No significant difference in GAPDH mRNA concentrations was seen between the 0.22-microm-filtered plasma samples and the ultracentrifuged plasma samples (P >0.05). In HCC patients, filtration with a 0.22 microm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) reduction in GAPDH mRNA concentration in plasma. Plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (P <0.0 5 for filtered plasma samples; P <0.005 for unfiltered plasma samples).
  
  
  CONCLUSIONS
  A substantial proportion of plasma mRNA species is particle-associated. In HCC patients, both circulating particle- and non-particle-associated plasma RNA are increased.
authors.affiliation: Institute of Molecular Oncology and Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region.
authors.forename: Enders K O
authors.initials: EK
authors.lastname: Ng
authors.forename: Nancy B Y
authors.initials: NB
authors.lastname: Tsui
authors.forename: Nicole Y L
authors.initials: NY
authors.lastname: Lam
authors.forename: Rossa W K
authors.initials: RW
authors.lastname: Chiu
authors.forename: Simon C H
authors.initials: SC
authors.lastname: Yu
authors.forename: S C Cesar
authors.initials: SC
authors.lastname: Wong
authors.forename: Elena S F
authors.initials: ES
authors.lastname: Lo
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Rainer
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Johnson
authors.forename: Y M Dennis
authors.initials: YM
authors.lastname: Lo
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000175:diagnosis*; D005374:Filtration; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood* / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood / Q000175:diagnosis*; D010949:Plasma; D012333:RNA, Messenger / Q000097:blood*; D012016:Reference Values; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014461:Ultracentrifugation
chemical_list: D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases