Method for isolation & accumulation of small RNAs for clinical marker development

A large part of gene regulation in eukaryotes takes place at the level of RNA-molecules. A group of endogenous regulatory RNA-molecules, the so-called microRNAs (miRNAs) appear to be involved in the regulation of about 30% of all human genes. These “small RNA” molecules (sRNA) also play a role in the development of diseases such as cancer, diabetes or neurological disorders. Disease-associated changes of miRNA expression patterns provide new prognostic and diagnostic opportunities and are qualified for the development of respective clinical markers.

To detect miRNA-derived biomarkers a whole-RNA purification from the sample has to be performed. For an identification of yet unknown miRNAs it is required to clone and sequence the purified sRNAs. In most cases miRNAs which are present in a sample at low concentrations are further depleted during the purification steps or even get lost, which renders in the results of the analysis non-representative and increases the risk to overlook potential biomarkers. The present invention is based on the finding, that the plant-viral protein ’Helper component proteinase’ (HC-Pro) can be used for a binding of sRNAs including miRNAs. Thus, HC-Pro can be used in order to enrich and to isolate sRNAs from samples. In addition a cupin protein was identified which clearly improves the binding of HC-Pro for sRNAs.

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