title: Cross Platform Standardisation of an Experimental Pipeline for Use in the Identification of Dysregulated Human Circulating MiRNAs.
abstract: BACKGROUND
  Micro RNAs (miRNAs) are a class of highly conserved small non-coding RNAs that play an important part in the post-transcriptional regulation of gene expression. A substantial number of miRNAs have been proposed as biomarkers for diseases. While reverse transcriptase Real-time PCR (RT-qPCR) is considered the gold standard for the evaluation and validation of miRNA biomarkers, small RNA sequencing is now routinely being adopted for the identification of dysregulated miRNAs. However, in many cases where putative miRNA biomarkers are identified using small RNA sequencing, they are not substantiated when RT-qPCR is used for validation. To date, there is a lack of consensus regarding optimal methodologies for miRNA detection, quantification and standardisation when different platform technologies are used.
  
  
  METHODS
  In this study we present an experimental pipeline that takes into consideration sample collection, processing, enrichment, and the subsequent comparative analysis of circulating small ribonucleic acids using small RNA sequencing and RT-qPCR.
  Initially, a panel of miRNAs dysregulated in circulating blood from breast cancer patients compared to healthy women were identified using small RNA sequencing. MiR-320a was identified as the most dysregulated miRNA between the two female cohorts. Total RNA and enriched small RNA populations (<30 bp) isolated from peripheral blood from the same female cohort samples were then tested for using a miR-320a RT-qPCR assay. When total RNA was analysed with this miR-320a RT-qPCR assay, a 2.3-fold decrease in expression levels was observed between blood samples from healthy controls and breast cancer patients. However, upon enrichment for the small RNA population and subsequent analysis of miR-320a using RT-qPCR, its dysregulation in breast cancer patients was more pronounced with an 8.89-fold decrease in miR-320a expression. We propose that the experimental pipeline outlined could serve as a robust approach for the identification and validation of small RNA biomarkers for disease.
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Helena
authors.initials: H
authors.lastname: Kelly
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Tim
authors.initials: T
authors.lastname: Downing
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Nina L
authors.initials: NL
authors.lastname: Tuite
authors.affiliation: Molecular Diagnostics Research Group (MDRG), School of Natural Sciences, National University of Ireland, Galway, Ireland; Biomedical Diagnostics Institute (BDI) Programme, National University of Ireland, Galway, Ireland.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Smith
authors.affiliation: Discipline of Surgery, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kerin
authors.affiliation: Discipline of Surgery, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Róisín M
authors.initials: RM
authors.lastname: Dwyer
authors.affiliation: Molecular Diagnostics Research Group (MDRG), School of Natural Sciences, National University of Ireland, Galway, Ireland; Biomedical Diagnostics Institute (BDI) Programme, National University of Ireland, Galway, Ireland.
authors.forename: Eoin
authors.initials: E
authors.lastname: Clancy
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Barry
authors.affiliation: Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Kate
authors.initials: K
authors.lastname: Reddington
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics; D016022:Case-Control Studies; D015331:Cohort Studies; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D017423:Sequence Analysis, RNA
chemical_list: D035683:MicroRNAs; D012334:RNA, Neoplasm