Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs

• Published in: Biochemia Medica 2021-02, Vol.31 (1), p.010901-148
• Main Authors: Sekovanić, Ankica, Dorotić, Adrijana, Jurasović, Jasna, Pašalić, Daria, Kovačić, Jelena, Stasenko, Sandra, Mioč, Tatjana, Piasek, Martina, Orct, Tatjana
• Format: Article
• Language: English
• Subjects: blood plasma; Circulating MicroRNA - blood; circulating microRNAs; Cross-Sectional Studies; epigenetics; Female; Fetal Blood - metabolism; Humans; Male; pre-amplification; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RT-PCR; Short Communications
• ISSN: 1330-0962; 1846-7482
• DOI: 10.11613/BM.2021.010901

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 presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. 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. Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (C ) 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). 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.