Pre-amplification as a method for improvement of quantitative rt-pcr analysis of circulating mirnas

Abstract

Introduction: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presuma-bly related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. Materials and methods: Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. Results: Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (CT ) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). Conclusion: Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification.