Next Article in Journal
Comparative Genomic Analysis of Transgenic Poplar Dwarf Mutant Reveals Numerous Differentially Expressed Genes Involved in Energy Flow
Previous Article in Journal
Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities
Article Menu
Issue 9 (September) cover image

Export Article

Open AccessShort Communication
Int. J. Mol. Sci. 2014, 15(9), 15592-15602; doi:10.3390/ijms150915592

Comprehensive Luciferase-Based Reporter Gene Assay Reveals Previously Masked Up-Regulatory Effects of miRNAs

Molecular Medicine Group, Robarts Research Institute, Western University, London, ON N6A 5B7, Canada
Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 3K7, Canada
Department of Pathology, Western University, London, ON N6A 3K7, Canada
These authors contributed equally to this work.
Author to whom correspondence should be addressed.
Received: 11 August 2014 / Revised: 25 August 2014 / Accepted: 28 August 2014 / Published: 3 September 2014
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
View Full-Text   |   Download PDF [2033 KB, uploaded 3 September 2014]   |  


MicroRNAs (miRNAs) are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region) target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs. View Full-Text
Keywords: reporter gene assay; miRNA; normalization; NEFL (neurofilament) reporter gene assay; miRNA; normalization; NEFL (neurofilament)

Figure 1

This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Campos-Melo, D.; Droppelmann, C.A.; Volkening, K.; Strong, M.J. Comprehensive Luciferase-Based Reporter Gene Assay Reveals Previously Masked Up-Regulatory Effects of miRNAs. Int. J. Mol. Sci. 2014, 15, 15592-15602.

Show more citation formats Show less citations formats

Related Articles

Article Metrics

Article Access Statistics



[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top