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Int. J. Mol. Sci. 2018, 19(10), 2858; https://doi.org/10.3390/ijms19102858

Identification of Endogenous Control miRNAs for RT-qPCR in T-Cell Acute Lymphoblastic Leukemia

1
Institute of Human Genetics, Polish Academy of Sciences, 60-479 Poznań, Poland
2
Department, Silesian University of Technology, 44-100 Gliwice, Poland
*
Author to whom correspondence should be addressed.
Received: 6 August 2018 / Revised: 12 September 2018 / Accepted: 18 September 2018 / Published: 20 September 2018
(This article belongs to the Special Issue MicroRNA as Biomarkers in Cancer Diagnostics and Therapy)
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Abstract

Optimal endogenous controls enable reliable normalization of microRNA (miRNA) expression in reverse-transcription quantitative PCR (RT-qPCR). This is particularly important when miRNAs are considered as candidate diagnostic or prognostic biomarkers. Universal endogenous controls are lacking, thus candidate normalizers must be evaluated individually for each experiment. Here we present a strategy that we applied to the identification of optimal control miRNAs for RT-qPCR profiling of miRNA expression in T-cell acute lymphoblastic leukemia (T-ALL) and in normal cells of T-lineage. First, using NormFinder for an iterative analysis of miRNA stability in our miRNA-seq data, we established the number of control miRNAs to be used in RT-qPCR. Then, we identified optimal control miRNAs by a comprehensive analysis of miRNA stability in miRNA-seq data and in RT-qPCR by analysis of RT-qPCR amplification efficiency and expression across a variety of T-lineage samples and T-ALL cell line culture conditions. We then showed the utility of the combination of three miRNAs as endogenous normalizers (hsa-miR-16-5p, hsa-miR-25-3p, and hsa-let-7a-5p). These miRNAs might serve as first-line candidate endogenous controls for RT-qPCR analysis of miRNAs in different types of T-lineage samples: T-ALL patient samples, T-ALL cell lines, normal immature thymocytes, and mature T-lymphocytes. The strategy we present is universal and can be transferred to other RT-qPCR experiments. View Full-Text
Keywords: miRNA (microRNA); T-cell acute lymphoblastic leukemia (T-ALL); normalization of miRNA expression in RT-qPCR; endogenous controls; reference genes; tissue analysis; cell lines miRNA (microRNA); T-cell acute lymphoblastic leukemia (T-ALL); normalization of miRNA expression in RT-qPCR; endogenous controls; reference genes; tissue analysis; cell lines
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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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Drobna, M.; Szarzyńska-Zawadzka, B.; Daca-Roszak, P.; Kosmalska, M.; Jaksik, R.; Witt, M.; Dawidowska, M. Identification of Endogenous Control miRNAs for RT-qPCR in T-Cell Acute Lymphoblastic Leukemia. Int. J. Mol. Sci. 2018, 19, 2858.

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