*4.6. Reverse Transcriptase Reaction*

Individual microRNAs were reverse transcribed into complementary DNA (cDNA) in a total reaction volume of 10 μL using microRNA-specific stem-loop RT primers, components of TaqMan MicroRNA Assays (Table 5), and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Branchburg, NJ, USA, no: 4366597). Reverse transcriptase reactions were performed with the following thermal cycling parameters: 30 min at 16 ◦C, 30 min at 42 ◦C, 5 min at 85 ◦C, and then held at 4 ◦C using a 7500 Real-Time PCR system (Applied Biosystems, Branchburg, NJ, USA).




**Table 5.** Characteristics of microRNAs involved in the study.

[+] A single strand of DNA sense (or positive (+)) if an RNA version of the same sequence is translated or translatable into protein. [−] Its complementary strand is called antisense (or negative (−) sense).

#### *4.7. Relative Quantification of microRNAs by Real-Time PCR*

3 μL of cDNA were mixed with specific TaqMan MGB probes and primers (TaqMan MicroRNA Assay, Applied Biosystems, Branchburg, NJ, USA), and the ingredients of the TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ, USA, no: 4318157). A total reaction volume was 15 μL. TaqMan PCR conditions were set up as described in the TaqMan guidelines for a 7500 Real-Time PCR system. All PCRs were performed in duplicates with the involvement of multiple negative controls such as NTC (water instead of cDNA sample), NAC (non-transcribed RNA samples), and genomic DNA (isolated from equal biological samples), which did not generate any signal during PCR reactions. The samples were considered positive if the amplification signal occurred at *Ct* < 40 (before the 40th threshold cycle).

The expression of particular microRNA was determined using the comparative Ct method [136] relative to normalization factor (geometric mean of two selected endogenous controls) [137]. Two non-coding small nucleolar RNAs (RNU58A and RNU38B) were optimal for qPCR data normalization in this setting. They demonstrated equal expression between children descending from normal and complicated pregnancies. RNU58A and RNU38B also served as positive controls for successful extraction of RNA from all samples and were used as internal controls for variations during the preparation of RNA, cDNA synthesis, and real-time PCR.

A reference sample, RNA fraction highly enriched for small RNAs isolated from the fetal part of one randomly selected placenta derived from gestation with normal course, was used throughout the study for relative quantification.
