Universally Label miRNAs from Any Source Including Plants

Source-independent Chemical miRNA Labeling

(A) Mammalian miRNAs can be labeled using either direct chemical labeling or commercially available enzymatic labeling methods. (B) Endogenous methylation of plant miRNAs at their 3’ ends inhibits labeling by the poly(A) polymerase-based kits. In contrast, plant miRNAs are efficiently labeled using only the Label IT® Kits due their direct chemical labeling technology.

Efficient Labeling and Sensitive Detection of Plant miRNAs and siRNAs

Small RNA samples containing both miRNAs and siRNAs were isolated from two strains of Arabidopsis thaliana, wild type and a dicer mutant strain (dcl1-9). In A. thaliana, dicer (Dcl1) is required for the production of miRNAs but not siRNAs. Thus, the abundance of miRNAs in the dcl1-9 mutant sample is expected to be dramatically reduced in wild type, while endogenous siRNA abundance should not be affected by the dicer mutation. To test this hypothesis, small RNA samples were labeled with Cy™5 using the Label IT® miRNA Labeling Kit and hybridized to duplicate Arabidopsis miRNA-specific microarrays containing custom Arabidopsis siRNA capture sequences (CombiMatrix). Corrected fluorescent signal of each feature was calculated as the ratio of feature fluorescence to mean background fluorescence. Corrected fluorescence signals for each target from duplicate hybridizations were averaged and the ratios of average wild type to average dicer mutant signals were plotted. Ratios of ~1 indicate no difference in miRNA or siRNA abundance between the two strains, however values >1 indicate a decrease in the level of the miRNA or siRNA in the dicer mutant. As expected, miRNA abundance in the dicer mutant was dramatically decreased while the siRNAs levels remained constant between the two strains.

Note: The data resulted from a collaboration between Xumei Chen, Ph.D., CombiMatrix, and Mirus Bio. We thank Dr. Chen for sharing the data prior to publication.

Sequence-independent Chemical miRNA Labeling

Synthetic miRNAs representing naturally occurring miRNAs with no Gs (miR-467a), no As (miR-328), no Cs (miR-206), and no Us (miR-214) were chemically labeled in triplicate with the Label IT® Cy™3 reagent, purified and spectrophotometrically analyzed to estimate labeling density (pmol Cy™3/μg RNA). Average labeling densities are plotted. Similar results were observed with Cy™5 labeling reactions (data not shown).