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

International Journal of Molecular Sciences
Monika DrobnaMałgorzata Dawidowska

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...Continue Reading

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Citations

Apr 26, 2019·International Journal of Molecular Sciences·Lorenzo F Sempere
Dec 19, 2018·Biomedical Papers of the Medical Faculty of the University Palacký, Olomouc, Czechoslovakia·David StejskalMarek Svestak
Jun 3, 2021·International Journal of Molecular Sciences·Katarzyna GębarowskaMonika Lejman

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Methods Mentioned

BETA
miRNA-seq
PCR

Software Mentioned

RefFinder
NormFinder
R
edgeR
Data Assist
Biostrings R package
BestKeeper
geNorm
Bowtie
IterativeStability

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