title: Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics.
abstract: BACKGROUND
  Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection.
  
  
  METHODS
  We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma.
  
  
  RESULTS
  We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 microm filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent.
  
  
  CONCLUSIONS
  Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite optimizing many aspects of plasma RNA detection, we were unable to reproducibly detect cancer-related transcripts. Our data suggest that measurement of circulating RNA may not be a good approach to early cancer diagnosis.
authors.affiliation: Division of Thoracic Surgery, School of Medicine, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Talal
authors.initials: T
authors.lastname: El-Hefnawy
authors.forename: Siva
authors.initials: S
authors.lastname: Raja
authors.forename: Lori
authors.initials: L
authors.lastname: Kelly
authors.forename: William L
authors.initials: WL
authors.lastname: Bigbee
authors.forename: John M
authors.initials: JM
authors.lastname: Kirkwood
authors.forename: James D
authors.initials: JD
authors.lastname: Luketich
authors.forename: Tony E
authors.initials: TE
authors.lastname: Godfrey
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm; D002498:Centrifugation; D004938:Esophageal Neoplasms / Q000097:blood / Q000175:diagnosis; D006801:Humans; D007381:Intermediate Filament Proteins / Q000097:blood; D053554:Keratin-20; D058965:MART-1 Antigen; D008545:Melanoma / Q000097:blood / Q000175:diagnosis; D051379:Mice; D014442:Monophenol Monooxygenase / Q000097:blood / Q000235:genetics; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics; D009369:Neoplasms / Q000097:blood / Q000175:diagnosis*; D010949:Plasma; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood; D012334:RNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D000951:Antigens, Neoplasm; D007381:Intermediate Filament Proteins; C507499:KRT20 protein, human; D053554:Keratin-20; C489975:Krt20 protein, mouse; D058965:MART-1 Antigen; C548667:MLANA protein, human; C548668:Mlana protein, mouse; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014442:Monophenol Monooxygenase; D012260:Ribonucleases