Publication details

Evaluation of miRNA detection methods for the analytical characteristics necessary for clinical utilization

Authors

KŘEPELKOVÁ Iveta BÁŤKOVÁ Tereza KŘÍŽÁKOVÁ Jitka DVORAKOVA B. CHALUPOVA L. MIKULÍK Robert SLABÝ Ondřej BARTOŠ Milan RUZICKA V.

Type Article in Periodical
Magazine / Source Biotechniques
MU Faculty or unit

Central European Institute of Technology

Citation
Web https://www.future-science.com/doi/pdf/10.2144/btn-2019-0021
Doi http://dx.doi.org/10.2144/btn-2019-0021
Keywords miREIA; miRNA biomarkers; miRNA enzyme immunoassay; RT-qPCR; SplintR-qPCR
Attached files
Description miRNAs are promising biomarkers but methods for their measurement are not clear. We therefore examined three miRNA detection technologies and considered the analytical characteristics essential for clinical utilization. TaqMan assays, SplintR-qPCR and miREIA were compared for their absolute quantification bias, conformity and robustness. Absolute concentrations of miR-142-5p, miR-23a-3p and miR-93-5p were measured with all three methods using 30 samples. Robustness was evaluated by measurement of miR-21-5p in five uniform experiments. Correlations were miRNA-specific, but we observed a different absolute concentration range in RT-qPCR (fmol/mu l) and methods evading the RT process (amol/mu l). Consistently, RT-less methods reported better robustness (CV 8-19%) than RT-qPCR (CV 39-50%). The calibration curve in TaqMan Advanced assay was influenced by dilution media. Methods avoiding RT seem to be a promising future alternative for miRNA measurement. METHOD SUMMARY Three miRNA detection technologies were compared: 1) RT-qPCR where the RT step was performed with either a specific (TaqMan miRNA assay) or universal (TaqMan Advanced assay) priming strategy; 2) miREIA technology, using hybridization and specific antibody to DNA/RNA hybrids and 3) SplintR-qPCR, which utilizes a hybridization and ligation step followed by qPCR.
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