title: The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis.
abstract: BACKGROUND
  To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer.
  
  
  METHODS
  Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis.
  
  
  RESULTS
  Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer.
  
  
  CONCLUSIONS
  Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression.
authors.affiliation: Molecular Oncology Laboratories, Department of Oncology, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK.
authors.forename: H E
authors.initials: HE
authors.lastname: Gee
authors.forename: F M
authors.initials: FM
authors.lastname: Buffa
authors.forename: C
authors.initials: C
authors.lastname: Camps
authors.forename: A
authors.initials: A
authors.lastname: Ramachandran
authors.forename: R
authors.initials: R
authors.lastname: Leek
authors.forename: M
authors.initials: M
authors.lastname: Taylor
authors.forename: M
authors.initials: M
authors.lastname: Patil
authors.forename: H
authors.initials: H
authors.lastname: Sheldon
authors.forename: G
authors.initials: G
authors.lastname: Betts
authors.forename: J
authors.initials: J
authors.lastname: Homer
authors.forename: C
authors.initials: C
authors.lastname: West
authors.forename: J
authors.initials: J
authors.lastname: Ragoussis
authors.forename: A L
authors.initials: AL
authors.lastname: Harris
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D002277:Carcinoma / Q000235:genetics; D002294:Carcinoma, Squamous Cell; D005260:Female; D006258:Head and Neck Neoplasms / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000032:analysis*; D018307:Neoplasms, Squamous Cell / Q000235:genetics; D011379:Prognosis; D020537:RNA, Small Nucleolar / Q000032:analysis / Q000502:physiology*; D000077195:Squamous Cell Carcinoma of Head and Neck
chemical_list: D035683:MicroRNAs; D020537:RNA, Small Nucleolar