Circulating MicroRNAs in cancer

It is believed that microRNAs have potential as circulating biomarkers of disease, however successful clinical implementation remains a challenge. This chapter highlights broad variations in approaches to microRNA analysis where whole blood, serum and plasma have each been employed as viable sources. Further discrepancies in approaches are seen in endogenous controls and extraction methods utilised. This has resulted in contradictory publications, even when the same microRNA is targeted in the same disease setting. Analysis of blood samples highlighted the impact of both collection method and storage, on the microRNA profile. Analysis of a panel of microRNAs across whole blood, serum and plasma originating from the same individual emphasised the impact of starting material on microRNA profile. This is a highly topical field of research with immense potential for translation into the clinical setting. Standardisation of sample harvesting, processing and analysis will be key to this translation. Methods of sample harvesting, preservation and analysis are outlined, with important mitigating factors highlighted.This material is based upon works supported by the Irish Cancer Society collaborative cancer research centre BREAST-PREDICT Grant CCRC13GAL and funding agency Breast Cancer Research.Peer reviewe

Circulating MicroRNAs in cancer

It is believed that microRNAs have potential as circulating biomarkers of disease, however successful clinical implementation remains a challenge. This chapter highlights broad variations in approaches to microRNA analysis where whole blood, serum and plasma have each been employed as viable sources. Further discrepancies in approaches are seen in endogenous controls and extraction methods utilised. This has resulted in contradictory publications, even when the same microRNA is targeted in the same disease setting. Analysis of blood samples highlighted the impact of both collection method and storage, on the microRNA profile. Analysis of a panel of microRNAs across whole blood, serum and plasma originating from the same individual emphasised the impact of starting material on microRNA profile. This is a highly topical field of research with immense potential for translation into the clinical setting. Standardisation of sample harvesting, processing and analysis will be key to this translation. Methods of sample harvesting, preservation and analysis are outlined, with important mitigating factors highlighted.This material is based upon works supported by the Irish Cancer Society collaborative cancer research centre BREAST-PREDICT Grant CCRC13GAL and funding agency Breast Cancer Research.Peer reviewe

Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer