ORIGINAL RESEARCH
Br. J. Biomed. Sci.
Influence of reference gene selection on miRNA quantification by RT-qPCR in human placental samples
Ankica Sekovanić 1
Tatjana Orct 1
Adrijana Dorotić 2
Daria Pašalić 3
Zorana Kljaković- Gašpić 1
Sandra Stasenko 4
Tatjana Mioč 4,5
Martina Piasek 1
Jasna Jurasović 1
1. Institute for Medical Research and Occupational Health, Zagreb, Croatia
2. University Hospital Sveti Duh, Zagreb, Croatia
3. University of Zagreb, School of Medicine, Zagreb, Croatia
4. Merkur University Hospital, Zagreb, Croatia
5. Poliklinika Harni, Zagreb, Croatia
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Abstract
ABSTRACT The gold standard for assessing expression of miRNAs, small molecules involved in numerous biological processes, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The reliability of RT-qPCR analysis results largely depends on accurate data normalization and the selection of an appropriate reference gene. This study evaluated the stability of five candidate reference genes—miR-525, miR-520c, SNORD48, miR-135b, and miR-143—in human placental samples. GeNorm, NormFinder, BestKeeper, and the delta Ct-method were used to evaluate gene expression stability. The effect of reference gene selection for normalization of target miRNAs (miR-1537, miR-190b, miR-16, miR-21, and miR-146a) expression in term placental samples from smokers and non-smokers was also investigated. All statistical tools identified miR-525, miR-520c, and SNORD48 as the three most stable reference genes, except for GeNorm, which recommendss the combination of the first two genes. All statistical tools consistently ranked the reference genes in the following order: miR-525>miR-520c>SNORD48. Normalization using SNORD48 and miR-525 produced comparable results for miR-21 expression in the placental samples, both in smokers and non-smokers, whereas normalization with miR-143 yielded markedly different outcomes compared to SNORD48 and miR-525. These findings highlight the considerable impact of reference gene selection on RT-qPCR results, emphasizing the importance of careful validation to avoid misinterpretation of gene expression data.
Summary
Keywords
MicroRNA, normalization, housekeeping genes, RT-PCR, placenta
Received
31 July 2025
Accepted
21 November 2025
Copyright
© 2025 Sekovanić, Orct, Dorotić, Pašalić, Kljaković- Gašpić, Stasenko, Mioč, Piasek and Jurasović. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Tatjana Orct
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