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Tips from the Bench: Nucleic Acid Labeling Tip

Efficiently Label Plant microRNA (miRNA) Using Label IT® Kits

Label Plant miRNAs for Expression Profiling

The Label IT miRNA Labeling Kits can be used to label any miRNA sample, including methylated plant miRNAs, making it an excellent choice for all miRNA microarray expression profiling applications. Endogenous plant miRNAs are methylated at their 3' ends (1). Many enzymatic miRNA labeling kits utilize a poly(A) polymerase tailing reaction to add nucleotides/labels to the 3' end of miRNAs, but poly(A) polymerase does not efficiently extend 3' methylated plant miRNAs (2) (Figure 1). In comparison, the Label IT Reagents efficiently label plant miRNAs (or any nucleic acid) because they utilize a chemical alkylation reaction to internally label miRNAs that do not interfere with downstream hybridization applications (Figure 2).
Endogenous Methylation of Plant microRNAs Inhibits Labeling with Poly(A) Polymerase
Figure 1. Endogenous Methylation of Plant microRNAs Inhibits Labeling with Poly(A) Polymerase.
 
Plant miRNA labeling
Figure 2. Sensitive Detection of Plant miRNAs using Label IT® miRNA Labeling Kits. 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. 
 
References:
  1. Methylation as a Crucial Step in Plant microRNA Biogenesis. Yu B., Z. Yang, J. Li, S. Minakhina, M. Yang, R.W. Padgett, R. Steward, and X. Chen. 2005.Science 307:932–935.
  2. Ambion's website: mirVana™ miRNA Labeling Kit - "Plant miRNAs cannot be labeled with this kit due to endogenous 3' methylation."
 
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