Direct quantification of 3′ terminal 2′-O-methylation of small RNAs by RT-qPCR

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FIGURE 2.
FIGURE 2.

Quantification of miRNA methylation ratio by ΔCt method. (A,B) Stem–loop primer RT-qPCR (A) and poly(A)-tailed RT-qPCR (B) Ct values of different methylation ratio synthetic MIR168. By substituting Ct values in the formula, we calculated the methylation ratio for each synthetic sample (n = 6). (C) Accuracy of ΔCt method in detecting various 2′Ome levels of synthetic MIR168. (D) Stem–loop primer RT-qPCR and poly(A)-tailed RT-qPCR Ct values of MIR168 in Arabidopsis thaliana, synthetic 2′Ome (methylated), and unmethylated MIR168 (n = 9). (E) Quantification of the methylation ratio of MIR168 in Arabidopsis thaliana by ΔCt method. The detected percentage of 2′Ome in MIR168 is indicated on the top of the column. Data are presented as Mean ± SE (n = 3).

This Article

  1. Published in Advance August 3, 2018, doi: 10.1261/rna.065144.117 RNA 24: 1520-1529

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