Document Type

Article

Publication Date

4-2013

MeSH Terms

Breast Neoplasms, Biomarkers

Subject: LCSH

Breast--Cancer

Disciplines

Biology | Forensic Science and Technology

Abstract

MicroRNAs (miRNAs) have been found to be stable in the circulation of cancer patients raising their potential as circulating biomarkers of disease. The specific source and role, however, of miRNAs in the circulation is unknown and requires elucidation to determine their true potential. In this study, along with primary tissue explants and primary stromal cells, three breast cancer cell lines were employed, including T47D, MDA-MB-231 and SK-BR-3. Tissue explants were harvested in theatre, with informed patient consent, and included tumour, tumour associated normal, and diseased lymph node samples. Cell-conditioned media containing all factors secreted by the cells were harvested. MiRNAs were extracted from samples using 5 different extraction techniques including the blood protocol, RNeasy® (Qiagen), miRNeasy®mini kit (Qiagen), mirVana™ isolation kit (Ambion) and RNAqueous® kit (Ambion). MiRNAs were successfully isolated from all media samples collected from cell lines, primary cells and fresh tissue explants. However, there was remarkable variation in yield depending on the extraction method used. Aliquots of the same samples were extracted, revealing the two column extraction protocol of the mirVana® miRNA isolation kit to be the most suitable approach. A range of miRNAs, including miR-16, miR-195, miR-497 and miR-10b, were successfully amplified. While miR-16 and miR-195 were detected in media from both cell lines and tissue explants, miR-497 and miR-10b were only detected in secretions from whole tissue explants. The ability to achieve reliable and reproducible miRNA yields from cell-conditioned media is vital for the successful amplification of miRNAs by RQ-PCR.

Comments

This is the authors' revised manuscript, accepted for publication in MicroRNA. The final publication is available at http://www.ingentaconnect.com/content/ben/mirna/2013/00000002/00000001/art00003.

Publisher Citation

Glynn, C. L., Khan, S., Kerin, M. J., & Dwyer, R. M. (2013). Isolation of secreted microRNAs (miRNAs) from cell-conditioned media. MicroRNA, 2(1), 14-19.

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