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Identification of Endogenous Controls for Use in miRNA Quantification in Human Cancer Cell Lines.

BACKGROUND
miRNAs play important roles in multiple biological processes, and deregulation has been linked to several human diseases, including cancer. Studying changes in miRNA expression in cancer development is commonly performed in vitro in human cancer cell lines using quantitative polymerase chain reaction (qPCR), a method requiring the use of a robust reference gene that displays stable expression across all samples.


MATERIALS AND METHODS
Using the NormFinder software, a selection of commonly used endogeneous controls and miRNAs were tested in six human cancer cell lines to identify for the most suitable gene for use as a reference.


RESULTS
The frequently used endogenous control U6B small nuclear RNA (RNU6B) was found to be an unsuitable reference for normalization. The most suitable single endogeneous control identified was miR-25-3p, whereas the best combination of two endogeneous controls was miR-25-3p and miR-93-5p.


CONCLUSION
We identified a single and a pair of miRNAs suitable for use as endogenous controls when performing qPCR-based miRNA expression analyses in human cancer cell lines.