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Updated: Jun 2, 2026
RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs
Published on: July 11, 2020
Northern blotting analysis of microRNAs, their precursors and RNA interference triggers
Edyta Koscianska1, Julia Starega-Roslan, Lukasz J Sznajder
1Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str,, 61-704 Poznan, Poland.
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.
Insights
High-resolution northern blotting effectively characterizes microRNA (miRNA) length variants and RNA interference (RNAi) reagents. This method complements deep sequencing for analyzing miRNA biogenesis and reagent quality.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) exhibit end-heterogeneity due to imprecise processing and nucleotide additions.
- Deep sequencing reveals numerous miRNA variants, including minor ones of questionable functional significance.
- RNA interference (RNAi) reagents face similar end-heterogeneity issues during cellular processing.
Purpose of the Study:
- To investigate the end-heterogeneity of endogenous and exogenous RNAs.
- To assess the utility of high-resolution northern blotting for miRNA and RNAi reagent analysis.
- To compare northern blotting results with miRNA deep sequencing data.
Main Methods:
- High-resolution northern blotting was employed to analyze endogenous and exogenous RNAs (20-70 nt).
- Northern blotting results were validated against miRNA deep sequencing data.
- The study focused on characterizing length variants and heterogeneity.
Main Results:
- High-resolution northern blotting accurately quantifies major length variants of abundant endogenous miRNAs.
- The technique effectively characterizes the length heterogeneity of cellularly expressed RNAi reagents.
- Northern blotting provides a valuable complement to deep sequencing for miRNA analysis.
Conclusions:
- Enhanced high-resolution northern blotting is a valuable tool for miRNA discovery and characterization.
- It offers quantitative insights into miRNA length variants and RNAi reagent heterogeneity.
- This method serves as a useful adjunct to existing miRNA detection and analysis techniques.
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