Isothermal chemiluminescent assay based on circular stand-displacement polymerization reaction amplification for cel-miRNA-39-3p determination in cell extracts

• Published in: International journal of biological macromolecules Vol. 182; pp. 987 - 992
• Main Authors: Solovjev, Anton M., Galkin, Ivan I., Pletjushkina, Olga Yu, Medvedko, Alexey V., Zhao, Shulin, Sakharov, Ivan Yu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2021
• Subjects: chemiluminescence; Circular strand-displacement polymerization reaction; cross reaction; detection limit; Enhanced chemiluminescence reaction; guanidines; Heterogeneous assay; Isothermal amplification; luminescent assay; MicroRNA; phenol; polymerization; Polyperoxidase amplification; silica; streptavidin; thiocyanates; Enhanced chemiluminescence reaction; MicroRNA; Heterogeneous assay; Isothermal amplification; Circular strand-displacement polymerization reaction; Polyperoxidase amplification
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2021.04.101

A sensitive and specific heterogeneous assay for quantitation of cel-miRNA-39-3p (miRNA-39) was constructed. To improve the assay sensitivity an amplification strategy based on the use of isothermal circular strand-displacement polymerization reaction (ICSDPR), polyperoxidase conjugated with streptavidin and enhanced chemiluminescence was used. The detection limit of the proposed assay was 4 × 10−13 M. The coefficient of variation (CV) for quantitation of miRNA-39 within the working range was below 8%. The study of cross-reactivity of different miRNAs including miRNA-39 demonstrated high specificity of the proposed assay. Comparison of the calibration curves of miRNA-39 dissolved in the buffer and the lysate of MCF-7 cells (prepared by lysis of the cells with phenol/guanidine thiocyanate mixture and purified using silica membrane spin column) has demonstrated a negligible matrix effect. The proposed assay makes it possible to estimate the yield of purification of miRNAs from cells, which is necessary for the quantitative calculation of the intracellular content of miRNAs measured with the isothermal assay coupled with ICSDPR. •Specific microplate-based assay for detection of cel-miRNA-39 was developed.•The assay was based on using ICSDPR, polyperoxidase and enhanced chemiluminescence.•The detection limit of the proposed assay was 400 fM.•The assay showed a negligible matrix effect upon detection of miRNA-39 purified from spiked samples of MCF-7 cells lysate.•The assay can be used for determination the yield of purification of miRNAs.