Quantification of Plant and Serum miRNAs

Taqman microRNA Assays for let-7d,g,i22 (link), let-7a, miR-16, MIR161, MIR2911, MIR168a, and C7 were obtained from Life Technologies (Grand Island, NY). For serum samples, total RNA equivalent to 10 μL of sera was used in each RT reaction. Of the 10 μL RT product, 1 μL was used for each triplicated qPCR reaction. To quantify miRNA levels in the plants, 10–30 mg of fresh plant material were ground to a fine powder in liquid nitrogen and then subjected to RNA isolation using the miRNEASY kit (Qiagen, Valencia, CA); 1 pmole of synthetic artificial miRNA C7 was spiked into the plant Qiazol lysate as an exogenous RNA control. The quantification result was normalized to the weight of starting plant material. qRT-PCR was performed using a Biorad CFX96 Real-Time PCR Detection System, and data were analyzed using Biorad CFX software9 (link). The 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. The quantification Ct values all fall in the linear range of taqman assays as determined by the synthetic microRNA standards.