RESUMEN
Heterogeneous and homogeneous-heterogeneous assays for the quantitation of hsa-miR-141-3p (miRNA-141) were constructed. Both microplate assays were based on the use of the isothermal circular strand-displacement polymerization reaction (ICSDPR), which was carried out in heterogeneous and homogeneous media, respectively. In addition, a streptavidin-polyperoxidase conjugate and enhanced chemiluminescence were used to increase the assay's sensitivity. A comparison of the developed assays showed that the sensitivity of the heterogeneous assay was higher than that of the homogeneous-heterogeneous assay. The detection limit values of the heterogeneous and homogeneous-heterogeneous assays were 51 fM and 10 pM, respectively. The amplification index for the ICSDPR used in the heterogeneous assay of miRNA-141 was 100. Using miRNAs of the miRNA-200 family, the high specificity of the assay was demonstrated. MiRNA-141 in human cultured cells was determined by the heterogeneous ICSDPR-assisted assay with chemiluminescence detection. To assess the purification yield of miRNAs from cellular lysates, the heterogeneous assay of miRNA-39 developed on the same platform was used. The intracellular content of miRNA-141 in Caco-2, HepG2, MCF-7 and HeLa was shown to be 3400, 1400, 1300 and 470 copies per cell, respectively.
Asunto(s)
MicroARNs , Técnicas de Amplificación de Ácido Nucleico , Células CACO-2 , Humanos , Límite de Detección , MicroARNs/genética , Polimerizacion , EstreptavidinaRESUMEN
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.
Asunto(s)
Mediciones Luminiscentes/métodos , MicroARNs/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Mediciones Luminiscentes/normas , Células MCF-7 , MicroARNs/análisis , Técnicas de Amplificación de Ácido Nucleico/normas , Peroxidasa/metabolismo , Sensibilidad y EspecificidadRESUMEN
Sensitive microplate-based chemiluminescent assay coupled with isothermal circular strand-displacement polymerization reaction (ICSDPR) for DNA detection was developed. The assay sensitivity was improved using a triple amplification strategy based on employment of ICSDPR, streptavidin-polyperoxidase conjugate and an enhanced chemiluminescent reaction. To increase the accuracy all stages of the assay (one-pot format) were carried out in the same microplate well. The proposed assay detected the target DNA in fM-pM ranges and distinguish target DNA from related mismatched DNAs, demonstrating high sensitivity and high selectivity. This format is highly suitable for the development of sensitive and high-throughput kits of nucleic acids which can be easily automated.