Quantification of Plant and Exosomal miRNAs

Taqman microRNA Assays for let-7dgi [24 (link)], miR-16, MIR161, MIR2911, MIR156a, MIR168a and artificial miRNA C7 were obtained from Life Technologies. Total RNA equivalent to 10 μL of sera or 8 μL of urine were used in each reverse transcription (RT) reaction. Of the 10 μL RT product, 0.5 μL was used for each triplicated quantitative polymerase chain reaction (PCR). To quantify miRNA levels in herbs and flowers, 10 mg of dried plant material were ground to fine powder in liquid nitrogen and then subjected to RNA isolation using the miRNEASY kit (Qiagen); 1 pmol of synthetic MIR161 was spiked into the plant Qiazol lysate as an exogenous RNA control. qRT-PCR was performed using a Biorad CFX96 Real-Time PCR Detection System, and data were analyzed using Biorad CFX software. Delta-Delta-Ct method was used to calculate relative levels of miRNAs. Absolute concentrations of miRNAs were calculated based on standard curves obtained from serial dilutions of synthetic miRNAs. To verify the fidelity of Taqman microRNA assay kit for MIR2911, the qPCR product was agarose gel-purified and subcloned into pGEM-T Easy vector (Promega) and sequenced [25 (link)].