Quantification of miRNA Levels in Arabidopsis

TaqMan miRNA Assays for let-7dgi [32 (link)], C7, amiR-RICE, mmu-miR146a, MIR2911, and MIR168a were obtained from Life Technologies (Carlsbad, CA). Total RNA isolated was used in each reverse transcription (RT) reaction, as previously described [14 (link)]. To quantify miRNA levels in Arabidopsis, plant shoot material was ground to a fine powder in liquid nitrogen and then 10–20 mg was subjected to RNA isolation using the miRNeasy kit; 50 fmol of synthetic C7 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 the relative levels of miRNAs. Absolute concentrations of miRNAs were calculated based on standard curves obtained from serial dilutions of synthetic miRNAs [14 (link)] (Additional file 1: Figure S1).