Characterizing multiple exogenous and endogenous small RNA populations in parallel with subfemtomolar sensitivity...

Here we present a simple and inexpensive gel-shift assay for the detection and quantification of small RNAs. The assay is at least 5-10 times more sensitive than a conventional Northern, and is highly scalable. Total RNA is first size purified to enrich the desired size range, phosphatase treated, and then radiolabeled to high specific activity using polynucleotide kinase. The resulting RNA stock is then hybridized to an excess of biotinylated DNA probe oligonucleotide, prior to mixing with streptavidin and loading on a native gel. The amount of supershifted material was proportional to the amount of labeled target RNA in the sample. We applied this method to verify sequencing data originally obtained from a four-point comparison study on the effect of endogenous expression of HC-Pro on Y-satellite/cucumber mosaic virus infection in tobacco plants. The results of the streptavidin gel-shift assay were consistent with the concentrations of small RNA infected plants inferred by our original cloning data, and rapidly provided information about the relative concentration of a number of viral and endogenous small RNAs. Further straightforward improvements to this simple methodology might be expected to improve the methods sensitivity by as much as another 10-fold.