RESUMO
Developing molecular diagnostics in resource-poor settings is challenging. As such, we purpose-built a novel bridging flocculation assay for qualitative evaluation of isothermally amplified DNA by naked eye. The flocculation assay was dependent on pH, DNA polymer amounts and lengths. The method was first applied to the rapid and sensitive detection of important plant pathogens and subsequently extended to other pathogens across the animal kingdom to demonstrate the wide applications of our approach.
Assuntos
DNA/análise , Fusarium/genética , HIV-1/genética , Vírus da Influenza A Subtipo H1N1/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Pseudomonas syringae/genética , Animais , Arabidopsis/microbiologia , Bovinos , DNA/economia , Floculação , Fusarium/isolamento & purificação , HIV-1/isolamento & purificação , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Células Jurkat , Técnicas de Amplificação de Ácido Nucleico/economia , Pseudomonas syringae/isolamento & purificação , Microextração em Fase Sólida/métodosRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs of â¼20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located â¼2 kb upstream of the 5' stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.