title: Identification of stably expressed reference small non-coding RNAs for microRNA quantification in high-grade serous ovarian carcinoma tissues.
abstract: MicroRNAs (miRNAs) belong to a family of small non-coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high-grade serous ovarian carcinoma (HGS-OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS-OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR-92a-3p, let-7a-5p, SNORD61, SNORD72, SNORD68, miR-103a-3p, miR-423-3p, miR-191-5p, miR-16-5p) were analysed on a total of 75 HGS-OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS-OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR-191-5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS-OvCa and normal controls, including the first time both the normal tissues supposed to be HGS-OvCa progenitors. In addition, we recommend miR-191-5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS-OvCa tissues.
authors.affiliation: Division of Obstetrics and Gynecology, ASST Spedali Civili di Brescia, Brescia, Italy.
authors.forename: Eliana
authors.initials: E
authors.lastname: Bignotti
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.|Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Stefano
authors.initials: S
authors.lastname: Calza
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Renata A
authors.initials: RA
authors.lastname: Tassi
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Zanotti
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Bandiera
authors.affiliation: Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Sartori
authors.affiliation: Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Franco E
authors.initials: FE
authors.lastname: Odicino
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Ravaggi
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.|Doctorate School of Molecular and Translational Medicine, University of Milan, Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Todeschini
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Romani
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D003198:Computer Simulation; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D018297:Neoplasms, Cystic, Mucinous, and Serous / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics*; D058727:RNA, Small Untranslated / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D012984:Software
chemical_list: D035683:MicroRNAs; D058727:RNA, Small Untranslated
keywords: 
  qPCR
  ; HGS-OvCa; endogenous reference; normalization; sncRNA