engleski

Study of preamplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs in maternal and cord blood plasma

Circulating extracellular microRNAs (miRNAs) in the serum or plasma has generated great interest as a promising biomarker for diverse pathological conditions - changes in miRNA expression in body fluids develop earlier than conventional biomarkers. Their expression in mother-newborn pairs may signal uterine exposure to environmental pollutants, including tobacco smoke (TS) and toxic metals, and the role of miRNAs in fetal programming remains largely unstudied. Assessment of extracellular miRNAs in plasma is limited and remains challenging due to their low amounts in circulation, small size and the lack of reference miRNA. Preamplification of the miRNA’s cDNA can be used to facilitate reliable quantification. Within our research, we assess daily exposure to main toxic metals and maternal susceptibility as factors of developmental origins of health and disease. Preliminary results on candidate miRNAs indicated aberrant expression of miR-21 and miR- 146a in maternal plasma related to TS exposure, whereas miR-1537 and miR-190b were not expressed or had a threshold cycle (Ct) value >35 in 95% and 50% of analyzed samples. We further optimized the protocol for circulating miRNA analysis (by Qiagen miRNA assays) and tested the preamplification step to increase the sensitivity of qPCR analysis and the number of detectable miRNAs in 19 paired maternal and umbilical cord blood plasma samples. By using the ∆∆Ct method of relative quantification, we analyzed fold change for miRNA expression in the samples from healthy smoking (n=10) and non-smoking (n=9) postpartum women collected immediately after delivery. Preamplification facilitated the detection of all assayed candidate miRNAs and yielded a mean Ct improvement of 6.7±1.1 (p<0.01) compared with Ct values derived from the direct qPCR method. Values of ∆Ct between these methods were highly correlated (r>0.9) for miR-16, miR-21 and miR- 146a (whose Ct values by direct qPCR were already <35). Moreover, fold changes for miR-1537 and miR-190b in cord blood plasma were 3.5 and 1.6 indicating their upregulation related to TS exposure. Our results confirm that preamplification is a useful and reliable procedure to facilitate extracellular miRNA expression detection in maternal and cord blood plasma.

candidate micro RNAs ; maternal blood plasma ; cord blood plasma ; quantitative RT-PCR ; tobacco smoke exposure

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