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

Anna Lindelov 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.
Original languageEnglish
Article number896
JournalInternational Journal of Molecular Sciences
Volume17
Issue number6
Number of pages8
ISSN1661-6596
DOIs
Publication statusPublished - 2016
Externally publishedYes

Cite this

Vestergaard, Anna Lindelov ; Blankestijn, Maaike ; Stahl, Jonathan Lucien ; Pallesen, Emil Marek Heymans ; Bang-Berthelsen, Claus Heiner ; Pociot, Flemming ; Novotny, Guy Wayne ; Lundh, Morten ; Mandrup-Poulsen, Thomas. / A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells. In: International Journal of Molecular Sciences . 2016 ; Vol. 17, No. 6.
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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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A Systematic Comparison of Purification and Normalization Protocols for Quantitative MicroRNA Expressional Profiling in Insulin-Producing Cells. / Vestergaard, Anna Lindelov; Blankestijn, Maaike; Stahl, Jonathan Lucien; Pallesen, Emil Marek Heymans; Bang-Berthelsen, Claus Heiner; Pociot, Flemming; Novotny, Guy Wayne; Lundh, Morten; Mandrup-Poulsen, Thomas.

In: International Journal of Molecular Sciences , Vol. 17, No. 6, 896, 2016.

Research output: Contribution to journalJournal articleResearchpeer-review

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AU - Vestergaard, Anna Lindelov

AU - Blankestijn, Maaike

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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JO - International Journal of Molecular Sciences (Online)

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