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

Anna Lindeløv Vestergaard; Maaike Blankestijn; Jonathan Lucien Stahl; Emil Marek Heymans Pallesen; Claus Heiner Bang-Berthelsen; Flemming Pociot; Guy Wayne Novotny; Morten Lundh; Thomas Mandrup-Poulsen

doi:10.3390/ijms17060896

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

Anna Lindeløv Vestergaard, Maaike Blankestijn, Jonathan Lucien Stahl, Emil Marek Heymans Pallesen, Claus Heiner Bang-Berthelsen, Flemming Pociot, Guy Wayne Novotny, Morten Lundh, Thomas Mandrup-Poulsen

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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.

Originalsprog	Engelsk
Artikelnummer	896
Tidsskrift	International Journal of Molecular Sciences (Online)
Vol/bind	17
Udgave nummer	6
Antal sider	8
ISSN	1661-6596
DOI	https://doi.org/10.3390/ijms17060896
Status	Udgivet - 7 jun. 2016

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10.3390/ijms17060896Licens: CC BY

A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing CellsForlagets udgivne version, 1,74 MBLicens: CC BY

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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. (2016). A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells. International Journal of Molecular Sciences (Online), 17(6), Artikel 896. https://doi.org/10.3390/ijms17060896

A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells. / Vestergaard, Anna Lindeløv; Blankestijn, Maaike; Stahl, Jonathan Lucien et al.
I: International Journal of Molecular Sciences (Online), Bind 17, Nr. 6, 896, 07.06.2016.

Publikation: Bidrag til tidsskrift › Kommentar/debat › Forskning › peer review

Vestergaard, AL, Blankestijn, M, Stahl, JL, Pallesen, EMH, Bang-Berthelsen, CH, Pociot, F, Novotny, GW, Lundh, M & Mandrup-Poulsen, T 2016, 'A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells', International Journal of Molecular Sciences (Online), bind 17, nr. 6, 896. https://doi.org/10.3390/ijms17060896

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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.",

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AU - Stahl, Jonathan Lucien

AU - Pallesen, Emil Marek Heymans

AU - Bang-Berthelsen, Claus Heiner

AU - Pociot, Flemming

AU - Novotny, Guy Wayne

AU - Lundh, Morten

AU - Mandrup-Poulsen, Thomas

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AB - 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.

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

Abstract

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