Determination of 2′-O-methylation status of miRNAs using miR-ID. (A) Quantification of 2′-OMe and 2′-OH forms of the same miRNA in a mixture. Synthetic let-7b miRNAs having 5′-p and either 2′-OH/3′-OH or 2′-OMe/3′-OH at their 3′-ends were used in this example. These miRNAs were mixed in five predefined proportions, with the total concentration of let-7b RNA constant at 20 pM. Each mixture was subjected to an miR-ID assay in which either Rnl1 or CLII was used in the circularization step, followed by the usual RTC and qPCR steps. For each assay, the Ct value was normalized and converted into a linear percentage, with the CLII assay results made equal to 100%. The Rnl1-based assay gives the concentration of 2′-OH form in the mixture, and normalization to the CLII- based results gives the relative percentage of the 2′-OH and 2′-OMe forms. (B) Identification of plant miRNAs having 2′-OMe or 2′-OH at their 3′-ends. miR-162a and miR-775 miRNAs were assayed in total RNA extracted from wild-type Arabidopsis thaliana plants (Ler) and hen1-1 mutants (Ler). In the wild type, all miRNAs have a 2′-OMe group at the 3′-end, whereas in the mutant, miRNAs are expected to be unmethylated. One hundred nanogram samples of total RNA were subjected to miR-ID assays in which either Rnl1 or CLII was used in the circularization step, followed by the usual RTC and qPCR steps. The Ct values obtained for the CLII-based assays were normalized to 1.00, and the relative signals obtained for Rnl1-based assays were calculated. Two biological replicates for both plant types represented by the gray and black columns are shown.
