Influence of Reference Gene Selection on miRNA Quantification by RT-qPCR in Human Placental Samples

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 recommends the combination of the first two genes. 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.