title: Direct saliva transcriptome analysis.
abstract: BACKGROUND
  Current standard operating procedures for salivary transcriptomic analysis require low temperatures and lengthy mRNA isolation, which substantially hamper its use in the clinic. We developed a streamlined, ambient-temperature processing, stabilization, and storage protocol for clinical analysis of salivary RNA.
  
  
  METHODS
  The direct saliva transcriptome analysis (DSTA) used cell-free saliva supernatant instead of isolated mRNA for saliva transcriptomic detection, and all procedures, including processing, stabilization, and storage of saliva samples, were performed at ambient temperature without a stabilizing reagent. We evaluated this streamlined protocol by comparing the mRNA expression levels of 3 saliva internal reference genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH); actin, beta (ACTB); and ribosomal protein S9 (RPS9)] to levels measured with standard procedures, and detecting the variation of their expression levels under long-term ambient temperature storage. The clinical utility of DSTA was assessed by use of 7 oral cancer salivary mRNA biomarkers in a clinical study.
  
  
  RESULTS
  Each saliva internal reference gene mRNA showed similar expression levels when assayed by the DSTA or standard procedures, and remained stable under ambient temperature storage for at least 10 weeks without significant degradation (P = 0.918, 0.288, and 0.242 for GAPDH, ACTB, and RPS9, respectively). Compared with standard procedures, the performance characteristics of oral cancer salivary transcriptomic markers were retained as assayed by DSTA after 10 weeks of storage at ambient temperature. These results indicate that the DSTA is a suitable alternative method for saliva transcriptomic analysis and is feasible for use in clinical cancer research applications.
  
  
  CONCLUSIONS
  The streamlined DSTA protocol can impact the saliva-handling method and improve the standard operating procedures for clinical saliva transcriptomic diagnostics.
authors.affiliation: School of Dentistry and Dental Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Yu-Hsiang
authors.initials: YH
authors.lastname: Lee
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Jean K
authors.initials: JK
authors.lastname: Reiss
authors.forename: Xinmin
authors.initials: X
authors.lastname: Yan
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: David
authors.initials: D
authors.lastname: Chia
authors.forename: David T W
authors.initials: DT
authors.lastname: Wong
mesh_terms: D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000032:analysis; D002294:Carcinoma, Squamous Cell / Q000737:chemistry; D005240:Feasibility Studies; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012463:Saliva / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger