Perfecta universal pcr primer

RNA from total pig plasma (n = 6) and plasma-derived EV (n = 4) was extracted using the miRNeasy kit for plasma (Qiagen) and Trizol LS, respectively. Both protocols were performed according to the manufacturer’s descriptions. To correct for isolation variability and to enable comparative analysis of total plasma and plasma EV, C. Elegans miRNA-39 (Quanta Biosciences) was added to the lysis buffer equalized to the starting amount of plasma. RNA quantity and quality were measured with the Nanodrop (NanoDrop Products) and the 2100 Small RNA Assay Bioanalyzer (Agilent). cDNA was synthesized with qScript™ microRNA cDNA Synthesis Kit (Quanta BioSciences), following the manufacturer’s protocol. Quantitative RT-PCR (qRT-PCR) was performed in 12.5 μl duplicate reactions with PerfeCTa SYBR Green SuperMix (BioSciences), the PerfeCTa Universal PCR Primer (Quanta Biosciences), and primers specific for miRNA-1, miRNA-21, miRNA-133b, miRNA-146a, miRNA-208b, and miRNA-499a.
The cycle number that exceeds the fluorescence threshold is the threshold cycle (Ct value). Ct values that exceeded 40 cycles were treated as Ct 42. At missing time points, the average of the other pigs in that specific group was used as the cycle number for that time point. Ct values were normalized by using the average Ct value of the spike-in miRNA.

Total RNA was extracted as previously described. The expression of miR-675-5p and miR-675-3p was quantified by quantitative RT-PCR. Single strand RNA is first polyadenylated by poly(A)polymerase before reverse transcription into cDNA using qScript RT with a proprietary adapter oligo(dT) primer. Using the kit: “the qScript microRNA cDNA Synthesis Kit” (Quanta Biosciences, VWR), following the manufacturer’s protocol. The step of amplification was carried out using the LightCycler technology, the PerfecCTa microRNA Assay, the PerfeCTa Universal PCR Primer and miRNA specific primers (Quanta Biosciences). SNORD44 serves as an internal control. The miRNA specific primers are listed in Additional file 1: Table S1.

Using 500 ng of total RNA, cDNA synthesis was performed with the qScript™ microRNA cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, USA) following the manufacturer’s instructions. The cDNA was diluted fivefold with nuclease-free water, and 1 µl was used as a template for qPCR. The amplification of miRNA was performed using the PerfeCTa^® microRNA assay primer, the PerfeCTa^® Universal PCR primer and the PerfeCTa^® SYBR^® Green SuperMix, Low ROX™ Kit (Quanta Biosciences, Gaithersburg, USA) as per the manufacturer’s instructions. qPCR was carried out on an Applied Biosystems 7500 FAST Real-Time PCR System. Relative miRNA levels were calculated using the ΔΔC_t method, and RNU6 was used as the miRNA PCR control.