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Relative and Absolute Expression Analysis of MicroRNAs Associated with Luminal A Breast Cancer– A Comparison

  • Original Article
  • Published:
Pathology & Oncology Research

Abstract

MicroRNAs, as small non-coding regulatory RNAs, play crucial roles in various aspects of breast cancer biology. They have prognostic and diagnostic value, which makes them very interesting molecules to investigate. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the gold standard method to analyse miRNA expression in breast cancer patients. This study investigated two RT-qPCR methods (absolute and relative) to determine the expression of ten miRNAs in whole blood samples obtained from luminal A breast cancer patients compared to healthy controls. Whole blood samples were collected from 38 luminal A breast cancer patients and 20 healthy controls in Paxgene blood RNA tubes. Total RNA was extracted and analysed by relative and absolute RT-qPCR. For relative RT-qPCR, miR-16 was used as an endogenous control. For absolute RT-qPCR, standard curves were generated using synthetic miRNA oligonucleotides to determine the absolute copy number of each miRNA. Of the ten miRNAs that were analysed, the absolute RT-qPCR method identified six miRNAs (miR-16, miR-145, miR-155, miR-451a, miR-21 and miR-486) that were upregulated and one miRNA (miR-195) that was downregulated. ROC curve and AUC analysis of the data found that the combination of three miRNAs (miR-145, miR-195 and miR-486) had the best diagnostic value for luminal A breast cancer with an AUC of 0.875, with 76% sensitivity and 81% specificity. On the other hand, the relative RT-qPCR method identified two miRNAs (miR-155 and miR-486) that were upregulated and miR-195, which was downregulated. Using this approach, the combination of three miRNAs (miR-155, miR-195 and miR-486) was showed to have an AUC of 0.657 with 65% sensitivity and 69% specificity. We conclude that miR-16 is not a suitable normalizer for the relative expression profiling of miRNAs in luminal A breast cancer patients. Compared to relative quantification, absolute quantification assay is a better method to determine the expression level of circulating miRNAs in Luminal A breast cancer.

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Authors and Affiliations

  1. Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science (NCBES), NUI Galway, Galway, Ireland

    Vahid Arabkari, Eoin Clancy & Terry J. Smith

  2. Discipline of Pathology, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, Ireland

    Vahid Arabkari

  3. Discipline of Surgery, School of Medicine, Lambe Institute for Translational Research, NUI Galway, Galway, Ireland

    Róisín M. Dwyer & Michael J. Kerin

  4. Clinical Research Facility and School of Mathematics, Statistics and Applied Mathematics, NUI Galway, Galway, Ireland

    Olga Kalinina, Emma Holian & John Newell

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Conceived and designed the experiments: TJS VA. Performed the experiments: VA. Analysed the data: VA EH OK JN. Contributed reagents/materials/analysis tools: TJS RMD MJK. Wrote the paper: VA EC TJS.

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Arabkari, V., Clancy, E., Dwyer, R.M. et al. Relative and Absolute Expression Analysis of MicroRNAs Associated with Luminal A Breast Cancer– A Comparison. Pathol. Oncol. Res. 26, 833–844 (2020). https://doi.org/10.1007/s12253-019-00627-y

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