Northern blotting analysis of microRNAs, their precursors and RNA interference triggers.

BACKGROUND

Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide additions. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amounts and are unlikely to be of functional importance. All RNA interference (RNAi) technology reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics.

RESULTS

In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resolution northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technology reagents.

CONCLUSIONS

The conventional northern blotting enhanced to high resolution may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quantitative data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technology reagents expressed in cells.