In the present work, we describe the development of a chemiluminescent enzyme-linked oligonucleotide assay coupled with mismatched catalytic hairpin assembly (mCHA) amplification for the quantitative determination of microRNA-155. To improve its sensitivity, a polymerase-free mCHA reaction was applied as an isothermal amplification method. The detection limit of the proposed assay was 400 fM. In addition, the high specificity of the assay was demonstrated. The proposed assay allowed assessment of the content of microRNA-155 in human cancer lines such as HepG2, Caco2, MCF7, and HeLa. The quantitation of microRNA-155 was performed after purification of short RNAs (less than 200 nt) from cell lysates since a high matrix effect was observed without this pre-treatment. The results of the quantitative determination of the microRNA content in cells were normalized using nematode microRNA-39, the concentration of which was determined using a heterogeneous assay developed by us using a strategy identical to that of the microRNA-155 assay.
Keywords: microRNA detection; mismatched catalytic hairpin assembly; polymerase-free isothermal amplification of nucleic acids.