title: MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer.
abstract: BACKGROUND
  MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer.
  
  
  METHODS
  We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa.
  
  
  RESULTS
  From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation).
  
  
  CONCLUSIONS
  We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.
authors.affiliation: Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Anne Haahr Mellergaard
authors.initials: AH
authors.lastname: Eriksen
authors.affiliation: Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Rikke Fredslund
authors.initials: RF
authors.lastname: Andersen
authors.affiliation: Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.|Department of Clinical Pathology, Roskilde Hospital, Roskilde, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Pallisgaard
authors.affiliation: Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Flemming Brandt
authors.initials: FB
authors.lastname: Sørensen
authors.affiliation: Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Jakobsen
authors.affiliation: Danish Colorectal Cancer Center South, Center of Clinical Excellence, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Torben Frøstrup
authors.initials: TF
authors.lastname: Hansen
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D000068240:Data Anonymization; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012004:Rectal Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D012015:Reference Standards
chemical_list: D014408:Biomarkers, Tumor; C546818:MIRN193 microRNA, human; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; C487931:mirnlet7 microRNA, human