title: An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years.
abstract: OBJECTIVE
  To detect non-viral mRNA in human plasma that has been frozen for three years using a new protocol.
  
  
  METHODS
  Plasma from 15 patients with colorectal cancer and 10 normal subjects was separated and frozen with Trizol at -80 degrees C for three years. As a control measure, plasma from 10 of the 15 patients was separated using the same protocol but no Trizol during storage. After three years, all samples were extracted using Trizol and RNeasy before the reverse transcriptase polymerase chain reaction was performed to detect non-viral beta catenin mRNA. In addition, extraction of three plasma samples by Trizol or RNeasy independently was carried out for comparison.
  
  
  RESULTS
  beta Catenin mRNA was detected in all 15 patient plasma samples and only one of the 10 normal subjects. In contrast, no beta catenin mRNA was found in the control and patient samples that were independently extracted by Trizol and RNeasy kit.
  
  
  CONCLUSIONS
  This new protocol is a reliable method for extracting non-viral mRNA from the plasma of patients with cancer after longterm storage for three years. Extractions using Trizol and RNeasy kits independently could not isolate mRNA with sufficient quantity and quality for detection.
authors.affiliation: Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region, China. cesar01@netvigator.com
authors.forename: S C C
authors.initials: SC
authors.lastname: Wong
authors.forename: E S F
authors.initials: ES
authors.lastname: Lo
authors.forename: M T
authors.initials: MT
authors.lastname: Cheung
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D002985:Clinical Protocols; D015179:Colorectal Neoplasms / Q000097:blood / Q000175:diagnosis*; D015925:Cryopreservation; D003598:Cytoskeletal Proteins / Q000235:genetics; D006801:Humans; D012333:RNA, Messenger / Q000097:blood / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000097:blood / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D015534:Trans-Activators / Q000235:genetics; D051176:beta Catenin
chemical_list: D014408:Biomarkers, Tumor; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011933:Reagent Kits, Diagnostic; D015534:Trans-Activators; D051176:beta Catenin