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Open AccessCommunication
Int. J. Mol. Sci. 2016, 17(6), 896; doi:10.3390/ijms17060896

A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells

1
Laboratory for Immuno-Endocrinology, Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark
2
Center for Non-coding RNA in Technology and Health, Department of Pediatrics, Herlev and Gentofte Hospital, 2730 Herlev, Denmark
3
Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark
*
Author to whom correspondence should be addressed.
Academic Editor: Y-h. Taguchi
Received: 15 April 2016 / Revised: 12 May 2016 / Accepted: 30 May 2016 / Published: 7 June 2016
(This article belongs to the Special Issue MicroRNA Regulation)
View Full-Text   |   Download PDF [1780 KB, uploaded 7 June 2016]   |  

Abstract

As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted. View Full-Text
Keywords: method; microRNA; purification; qPCR; quantification; normalization; pancreas method; microRNA; purification; qPCR; quantification; normalization; pancreas
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Vestergaard, A.L.; Blankestijn, M.; Stahl, J.L.; Pallesen, E.M.H.; Bang-Berthelsen, C.H.; Pociot, F.; Novotny, G.W.; Lundh, M.; Mandrup-Poulsen, T. A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells. Int. J. Mol. Sci. 2016, 17, 896.

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