1. Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity
- Author
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Macarena Perán, Houria Boulaiz, Cynthia Morata-Tarifa, Juan A. Marchal, Carmen Griñán-Lisón, María Ángel García, Manuel Picon-Ruiz, [Morata-Tarifa,C, Picon-Ruiz,M, Griñan-Lison,C, Boulaiz,H, Perán,M, Garcia,MA, Marchal,JA] Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada, Granada, Spain. [Morata-Tarifa,C, Marchal,JA] Biosanitary Institute of Granada (ibs.GRANADA), University Hospitals of Granada-University of Granada, Granada, Spain. [Morata-Tarifa,C, Picón-Ruiz,M] Braman Family Breast Cancer Institute, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, Florida, USA. [Boulaiz,H, Marchal,JA] Department of Human Anatomy and Embryology, University of Granada, Granada, Spain. [Perán,M] Department of Health Sciences, University of Jaén, Jaén, Spain. [Garcia,MA] Virgen de las Nieves University Hospital, Oncology Department, Oncology Unit, Granada, Spain., and This work was supported in part by grants from the Fundación Pública Andaluza Progreso y Salud, Consejería de Salud Junta de Andalucía in collaboration with JANNSSEN CILAG,S.A. (project numbers PI-0533-2014, PI-0441-2014), from the Ministerio de Economía y Competitividad (MINECO, FEDER funds, grant number MAT2015-62644-C2-2-R) from the Ministry of Economy and Competitiveness, Instituto de Salud Carlos III (FEDER funds, project DTS15/00174) and from the Chair 'Doctors Galera-Requena in cancer stem cell research'.
- Subjects
0301 basic medicine ,Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::RNA::RNA, Nuclear::RNA, Small Nuclear [Medical Subject Headings] ,Colorectal cancer ,medicine.disease_cause ,Bioinformatics ,0302 clinical medicine ,Anatomy::Cells::Cells, Cultured::Cell Line [Medical Subject Headings] ,Neoplasms ,ARN nucleolar pequeño ,Small nucleolar RNA ,MicroARNs ,Multidisciplinary ,Línea celular ,Diseases::Neoplasms::Neoplastic Processes::Carcinogenesis [Medical Subject Headings] ,Housekeeping gene ,Gene Expression Regulation, Neoplastic ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Epidemiologic Research Design::Reproducibility of Results [Medical Subject Headings] ,Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Computational Biology [Medical Subject Headings] ,030220 oncology & carcinogenesis ,Neoplasias de la mama ,Biología computacional ,Carcinogénesis ,Tumour heterogeneity ,Chemicals and Drugs::Biological Factors::Biomarkers [Medical Subject Headings] ,Computational biology ,Biology ,Real-Time Polymerase Chain Reaction ,Article ,03 medical and health sciences ,Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, Essential [Medical Subject Headings] ,Cell Line, Tumor ,microRNA ,Biomarkers, Tumor ,medicine ,Humans ,Neoplasias del colon ,Diseases::Neoplasms::Neoplasms by Site::Breast Neoplasms [Medical Subject Headings] ,Reacción en cadena en tiempo real de la polimerasa ,Reproducibilidad de resultados ,Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Therapeutic Uses::Antineoplastic Agents [Medical Subject Headings] ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Nucleic Acid Amplification Techniques::Polymerase Chain Reaction::Real-Time Polymerase Chain Reaction [Medical Subject Headings] ,Computational Biology ,Cancer ,Oncogenes ,medicine.disease ,Antineoplásicos ,Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Antisense Elements (Genetics)::RNA, Antisense::MicroRNAs [Medical Subject Headings] ,MicroRNAs ,030104 developmental biology ,Biomarcadores ,Cancer cell ,Diseases::Neoplasms::Neoplasms by Site::Digestive System Neoplasms::Gastrointestinal Neoplasms::Intestinal Neoplasms::Colorectal Neoplasms::Colonic Neoplasms [Medical Subject Headings] ,Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, Neoplasm::Oncogenes [Medical Subject Headings] ,Carcinogenesis ,Genes esenciales - Abstract
Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs (U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.
- Published
- 2017