Title:Isolation of Secreted microRNAs (miRNAs) from Cell-conditioned Media
VOLUME: 2 ISSUE: 1
Author(s):Claire L. Glynn, Sonja Khan, Michael J. Kerin and Roisin M. Dwyer
Affiliation:Discipline of Surgery, School of Medicine, Clinical Science Institute, National University of Ireland Galway, Costello Road, Galway, Ireland.
Keywords:Biomarker, breast cancer, cell culture media, circulation, extraction, microRNA, secretion.
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.
